α-Methylacyl-CoA Racemase from Mycobacterium tuberculosis-Detailed Kinetic and Structural Characterization of the Active Site.

α-Methylacyl-CoA racemase in M. tuberculosis (MCR) has an essential role in fatty acid metabolism and cholesterol utilization, contributing to the bacterium's survival and persistence. Understanding the enzymatic activity and structural features of MCR provides insights into its physiological and pathological significance and potential as a therapeutic target. Here, we report high-resolution crystal structures for wild-type MCR in a new crystal form (at 1.65 Å resolution) and for three active-site mutants, H126A, D156A and E241A, at 2.45, 1.64 and 1.85 Å resolutions, respectively. Our analysis of the new wild-type structure revealed a similar dimeric arrangement of MCR molecules to that previously reported and details of the catalytic site. The determination of the structures of these H126A, D156A and E241A mutants, along with their detailed kinetic analysis, has now allowed for a rigorous assessment of their catalytic properties. No significant change outside the enzymatic active site was observed in the three mutants, establishing that the diminution of catalytic activity is mainly attributable to disruption of the catalytic apparatus involving key hydrogen bonding and water-mediated interactions. The wild-type structure, together with detailed mutational and biochemical data, provide a basis for understanding the catalytic properties of this enzyme, which is important for the design of future anti-tuberculosis drug molecules.

Structural insights into the inhibitory mechanism of angiotensin-I-converting enzyme by the lactotripeptides IPP and VPP.

Human somatic angiotensin-1-converting enzyme (sACE) is composed of a catalytic N-(nACE) and C-domain (cACE) of similar size with different substrate specificities. It is involved in the regulation of blood pressure by converting angiotensin I to the vasoconstrictor angiotensin II and has been a major focus in the development of therapeutics for hypertension. Bioactive peptides from various sources, including milk, have been identified as natural ACE inhibitors. We report the structural basis for the role of two lacototripeptides, Val-Pro-Pro and Ile-Pro-Pro, in domain-specific inhibition of ACE using X-ray crystallography and kinetic analysis. The lactotripeptides have preference for nACE due to altered polar interactions distal to the catalytic zinc ion. Elucidating the mechanism of binding and domain selectivity of these peptides also provides important insights into the functional roles of ACE.

Recombinant protein production for structural and kinetic studies: A case study using M. tuberculosis α-methylacyl-CoA racemase (MCR).

Modern drug discovery is a target-driven approach in which a particular protein such as an enzyme is implicated in the disease process. Commonly, small-molecule drugs are identified using screening, rational design, and structural biology approaches. Drug screening, testing and optimization is typically conducted in vitro, and copious amounts of protein are required. The advent of recombinant DNA technologies has resulted in a rise in proteins purified by affinity techniques, typically by incorporating an "affinity tag" at the N- or C-terminus. Use of these tagged proteins and affinity techniques comes with a host of issues. This chapter describes the production of an untagged enzyme, α-methylacyl-CoA racemase (MCR) from Mycobacterium tuberculosis, using a recombinant E. coli system. Purification of the enzyme on a 100 mg scale using tandem anion-exchange chromatographies (DEAE-sepharose and RESOURCE-Q columns), and size-exclusion chromatographies is described. A modified protocol allowing the purification of cationic proteins is also described, based on tandem cation-exchange chromatographies (using CM-sepharose and RESOURCE-S columns) and size-exclusion chromatographies. The resulting MCR protein is suitable for biochemical and structural biology applications. The described protocols have wide applicability to the purification of other recombinant proteins and enzymes without using affinity chromatography.

Engineering Plastic Eating Enzymes Using Structural Biology.

Plastic pollution has emerged as a significant environmental concern in recent years and has prompted the exploration of innovative biotechnological solutions to mitigate plastic's negative impact. The discovery of enzymes capable of degrading specific types of plastics holds promise as a potential solution. However, challenges with efficiency, industrial scalability, and the diverse range of the plastic waste in question, have hindered their widespread application. Structural biology provides valuable insights into the intricate interactions between enzymes and plastic materials at an atomic level, and a deeper understanding of their underlying mechanisms is essential to harness their potential to address the mounting plastic waste crisis. This review article examines the current biochemical and biophysical methods that may facilitate the development of enzymes capable of degrading polyethylene terephthalate (PET), one of the most extensively used plastics. It also discusses the challenges that must be addressed before substantial advancements can be achieved in using these enzymes as a solution to the plastic pollution problem.

Molecular basis of selective amyloid-ß degrading enzymes in Alzheimer's disease.

The accumulation of the small 42-residue long peptide amyloid-ß (Aß) has been proposed as a major trigger for the development of Alzheimer's disease (AD). Within the brain, the concentration of Aß peptide is tightly controlled through production and clearance mechanisms. Substantial experimental evidence now shows that reduced levels of Aß clearance are present in individuals living with AD. This accumulation of Aß can lead to the formation of large aggregated amyloid plaques-one of two detectable hallmarks of the disease. Aß-degrading enzymes (ADEs) are major players in the clearance of Aß. Stimulating ADE activity or expression, in order to compensate for the decreased clearance in the AD phenotype, provides a promising therapeutic target. It has been reported in mice that upregulation of ADEs can reduce the levels of Aß peptide and amyloid plaques-in some cases, this led to improved cognitive function. Among several known ADEs, neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), insulin degrading enzyme (IDE) and angiotensin-1 converting enzyme (ACE) from the zinc metalloprotease family have been identified as important. These ADEs have the capacity to digest soluble Aß which, in turn, cannot form the toxic oligomeric species. While they are known for their amyloid degradation, they exhibit complexity through promiscuous nature and a broad range of substrates that they can degrade. This review highlights current structural and functional understanding of these key ADEs, giving some insight into the molecular interactions that leads to the hydrolysis of peptide substrates, the crucial tasks performed by them and the potential for therapeutic use in the future.

Crystal Structure of the Catalytic Domain of a Botulinum Neurotoxin Homologue from Enterococcus faecium: Potential Insights into Substrate Recognition.

Clostridium botulinum neurotoxins (BoNTs) are the most potent toxins known, causing the deadly disease botulism. They function through Zn2+-dependent endopeptidase cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, preventing vesicular fusion and subsequent neurotransmitter release from motor neurons. Several serotypes of BoNTs produced by Clostridium botulinum (BoNT/A-/G and/X) have been well-characterised over the years. However, a BoNT-like gene (homologue of BoNT) was recently identified in the non-clostridial species, Enterococcus faecium, which is the leading cause of hospital-acquired multi-drug resistant infections. Here, we report the crystal structure of the catalytic domain of a BoNT homologue from Enterococcus faecium (LC/En) at 2.0 Å resolution. Detailed structural analysis in comparison with the full-length BoNT/En AlphaFold2-predicted structure, LC/A (from BoNT/A), and LC/F (from BoNT/F) revealed putative subsites and exosites (including loops 1-5) involved in recognition of LC/En substrates. LC/En also appears to possess a conserved autoproteolytic cleavage site whose function is yet to be established.

A Molecular Analysis of the Aminopeptidase P-Related Domain of PID-5 from Caenorhabditis elegans.

A novel protein, PID-5, has been shown to be a requirement for germline immortality and has recently been implicated in RNA-induced epigenetic silencing in the Caenorhabditis elegans embryo. Importantly, it has been shown to contain both an eTudor and aminopeptidase P-related domain. However, the silencing mechanism has not yet been fully characterised. In this study, bioinformatic tools were used to compare pre-existing aminopeptidase P molecular structures to the AlphaFold2-predicted aminopeptidase P-related domain of PID-5 (PID-5 APP-RD). Structural homology, metal composition, inhibitor-bonding interactions, and the potential for dimerisation were critically assessed through computational techniques, including structural superimposition and protein-ligand docking. Results from this research suggest that the metallopeptidase-like domain shares high structural homology with known aminopeptidase P enzymes and possesses the canonical 'pita-bread fold'. However, the absence of conserved metal-coordinating residues indicates that only a single Zn2+ may be bound at the active site. The PID-5 APP-RD may form transient interactions with a known aminopeptidase P inhibitor and may therefore recognise substrates in a comparable way to the known structures. However, loss of key catalytic residues suggests the domain will be inactive. Further evidence suggests that heterodimerisation with C. elegans aminopeptidase P is feasible and therefore PID-5 is predicted to regulate proteolytic cleavage in the silencing pathway. PID-5 may interact with PID-2 to bring aminopeptidase P activity to the Z-granule, where it could influence WAGO-4 activity to ensure the balanced production of 22G-RNA signals for transgenerational silencing. Targeted experiments into APPs implicated in malaria and cancer are required in order to build upon the biological and therapeutic significance of this research.

A Comprehensive Structural Analysis of Clostridium botulinum Neurotoxin A Cell-Binding Domain from Different Subtypes.

Botulinum neurotoxins (BoNTs) cause flaccid neuromuscular paralysis by cleaving one of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. BoNTs display high affinity and specificity for neuromuscular junctions, making them one of the most potent neurotoxins known to date. There are seven serologically distinct BoNTs (serotypes BoNT/A to BoNT/G) which can be further divided into subtypes (e.g., BoNT/A1, BoNT/A2…) based on small changes in their amino acid sequence. Of these, BoNT/A1 and BoNT/B1 have been utilised to treat various diseases associated with spasticity and hypersecretion. There are potentially many more BoNT variants with differing toxicological profiles that may display other therapeutic benefits. This review is focused on the structural analysis of the cell-binding domain from BoNT/A1 to BoNT/A6 subtypes (HC/A1 to HC/A6), including features such as a ganglioside binding site (GBS), a dynamic loop, a synaptic vesicle glycoprotein 2 (SV2) binding site, a possible Lys-Cys/Cys-Cys bridge, and a hinge motion between the HCN and HCC subdomains. Characterising structural features across subtypes provides a better understanding of how the cell-binding domain functions and may aid the development of novel therapeutics.

Crystal Structures of the Clostridium botulinum Neurotoxin A6 Cell Binding Domain Alone and in Complex with GD1a Reveal Significant Conformational Flexibility.

Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at the neuromuscular junction. This activity has been exploited to treat a range of diseases associated with hypersecretion of neurotransmitters, with formulations of BoNT/A commercially available as therapeutics. Generally, BoNT activity is facilitated by three essential domains within the molecule, the cell binding domain (HC), the translocation domain (HN), and the catalytic domain (LC). The HC, which consists of an N-terminal (HCN) and a C-terminal (HCC) subdomain, is responsible for BoNT's high target specificity where it forms a dual-receptor complex with synaptic vesicle protein 2 (SV2) and a ganglioside receptor on the surface of motor neurons. In this study, we have determined the crystal structure of botulinum neurotoxin A6 cell binding domain (HC/A6) in complex with GD1a and describe the interactions involved in ganglioside binding. We also present a new crystal form of wild type HC/A6 (crystal form II) where a large 'hinge motion' between the HCN and HCC subdomains is observed. These structures, along with a comparison to the previously determined wild type crystal structure of HC/A6 (crystal form I), reveals the degree of conformational flexibility exhibited by HC/A6.

Structural basis for the inhibition of human angiotensin-1 converting enzyme by fosinoprilat.

Human angiotensin I-converting enzyme (ACE) has two isoforms, somatic ACE (sACE) and testis ACE (tACE). The functions of sACE are widespread, with its involvement in blood pressure regulation most extensively studied. sACE is composed of an N-domain (nACE) and a C-domain (cACE), both catalytically active but have significant structural differences, resulting in different substrate specificities. Even though ACE inhibitors are used clinically, they need much improvement because of serious side effects seen in patients (~ 25-30%) with long-term treatment due to nonselective inhibition of nACE and cACE. Investigation into the distinguishing structural features of each domain is therefore of vital importance for the development of domain-specific inhibitors with minimal side effects. Here, we report kinetic data and high-resolution crystal structures of both nACE (1.75 Å) and cACE (1.85 Å) in complex with fosinoprilat, a clinically used inhibitor. These structures allowed detailed analysis of the molecular features conferring domain selectivity by fosinoprilat. Particularly, altered hydrophobic interactions were observed to be a contributing factor. These experimental data contribute to improved understanding of the structural features that dictate ACE inhibitor domain selectivity, allowing further progress towards designing novel 2nd-generation domain-specific potent ACE inhibitors suitable for clinical administration, with a variety of potential future therapeutic benefits. DATABASE: The atomic coordinates and structure factors for nACE-fosinoprilat and cACE-fosinoprilat structures have been deposited with codes 7Z6Z and 7Z70, respectively, in the RCSB Protein Data Bank, www.pdb.org.

Structural Features of Clostridium botulinum Neurotoxin Subtype A2 Cell Binding Domain.

Botulinum neurotoxins (BoNT) are a group of clostridial toxins that cause the potentially fatal neuroparalytic disease botulism. Although highly toxic, BoNTs are utilized as therapeutics to treat a range of neuromuscular conditions. Several serotypes (BoNT/A-/G, /X) have been identified with vastly differing toxicological profiles. Each serotype can be further sub-categorised into subtypes due to subtle variations in their protein sequence. These minor changes have been attributed to differences in both the duration of action and potency for BoNT/A subtypes. BoNTs are composed of three domains-a cell-binding domain, a translocation domain, and a catalytic domain. In this paper, we present the crystal structures of the botulinum neurotoxin A2 cell binding domain, both alone and in complex with its receptor ganglioside GD1a at 1.63 and 2.10 Å, respectively. The analysis of these structures reveals a potential redox-dependent Lys-O-Cys bridge close to the ganglioside binding site and a hinge motion between the HCN and HCC subdomains. Furthermore, we make a detailed comparison with the previously reported HC/A2:SV2C structure for a comprehensive structural analysis of HC/A2 receptor binding.

Crystal Structures of Botulinum Neurotoxin Subtypes A4 and A5 Cell Binding Domains in Complex with Receptor Ganglioside.

Botulinum neurotoxins (BoNT) cause the potentially fatal neuroparalytic disease botulism that arises due to proteolysis of a SNARE protein. Each BoNT is comprised of three domains: a cell binding domain (HC), a translocation domain (HN), and a catalytic (Zn2+ endopeptidase) domain (LC). The HC is responsible for neuronal specificity by targeting both a protein and ganglioside receptor at the neuromuscular junction. Although highly toxic, some BoNTs are commercially available as therapeutics for the treatment of a range of neuromuscular conditions. Here we present the crystal structures of two BoNT cell binding domains, HC/A4 and HC/A5, in a complex with the oligosaccharide of ganglioside, GD1a and GM1b, respectively. These structures, along with a detailed comparison with the previously reported apo-structures, reveal the conformational changes that occur upon ganglioside binding and the interactions involved.

Probing the Requirements for Dual Angiotensin-Converting Enzyme C-Domain Selective/Neprilysin Inhibition.

Selective inhibition of the angiotensin-converting enzyme C-domain (cACE) and neprilysin (NEP), leaving the ACE N-domain (nACE) free to degrade bradykinin and other peptides, has the potential to provide the potent antihypertensive and cardioprotective benefits observed for nonselective dual ACE/NEP inhibitors, such as omapatrilat, without the increased risk of adverse effects. We have synthesized three 1-carboxy-3-phenylpropyl dipeptide inhibitors with nanomolar potency based on the previously reported C-domain selective ACE inhibitor lisinopril-tryptophan (LisW) to probe the structural requirements for potent dual cACE/NEP inhibition. Here we report the synthesis, enzyme kinetic data, and high-resolution crystal structures of these inhibitors bound to nACE and cACE, providing valuable insight into the factors driving potency and selectivity. Overall, these results highlight the importance of the interplay between the S1' and S2' subsites for ACE domain selectivity, providing guidance for future chemistry efforts toward the development of dual cACE/NEP inhibitors.

Molecular basis of C-mannosylation - a structural perspective.

The structural and functional diversity of proteins can be enhanced by numerous post-translational modifications. C-mannosylation is a rare form of glycosylation consisting of a single alpha or beta D-mannopyranose forming a carbon-carbon bond with the pyrrole ring of a tryptophan residue. Despite first being discovered in 1994, C-mannosylation is still poorly understood and 3D structures are available for only a fraction of the total predicted C-mannosylated proteins. Here, we present the first comprehensive review of C-mannosylated protein structures by analysing the data for all 10 proteins with C-mannosylation/s deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). We analysed in detail the WXXW/WXXWXXW consensus motif and the highly conserved pair of arginine residues in thrombospondin type 1 repeat C-mannosylation sites or homologous arginine residues in other domains. Furthermore, we identified a conserved PXP sequence C-terminal of the C-mannosylation site. The PXP motif forms a tight turn region in the polypeptide chain and its universal conservation in C-mannosylated protein is worthy of further experimental study. The stabilization of C-mannopyranosyl groups was demonstrated through hydrogen bonding with arginine and other charged or polar amino acids. Where possible, the structural findings were linked to other functional studies demonstrating the role of C-mannosylation in protein stability, secretion or function. With the current technological advances in structural biology, we hope to see more progress in the study of C-mannosylation that may correspond to discoveries of novel C-mannosylation pathways and functions with implications for human health and biotechnology.

Angiotensin-converting enzyme open for business: structural insights into the subdomain dynamics.

Angiotensin-1-converting enzyme (ACE) is a key enzyme in the renin-angiotensin-aldosterone and kinin systems where it cleaves angiotensin I and bradykinin peptides, respectively. However, ACE also participates in numerous other physiological functions, can hydrolyse many peptide substrates and has various exo- and endopeptidase activities. ACE achieves this complexity by containing two homologous catalytic domains (N- and C-domains), which exhibit different substrate specificities. Here, we present the first open conformation structures of ACE N-domain and a unique closed C-domain structure (2.0 Å) where the C terminus of a symmetry-related molecule is observed inserted into the active-site cavity and binding to the zinc ion. The open native N-domain structure (1.85 Å) enables comparison with ACE2, a homologue previously observed in open and closed states. An open S2 _S'-mutant N-domain structure (2.80 Å) includes mutated residues in the S2 and S' subsites that effect ligand binding, but are distal to the binding site. Analysis of these structures provides important insights into how structural features of the ACE domains are able to accommodate the wide variety of substrates and allow different peptidase activities. DATABASE: The atomic coordinates and structure factors for Open nACE, Open S2_S'-nACE and Native G13-cACE structures have been deposited with codes 6ZPQ, 6ZPT and 6ZPU, respectively, in the RCSB Protein Data Bank, www.pdb.org.

ACE2 and ACE: structure-based insights into mechanism, regulation and receptor recognition by SARS-CoV.

Angiotensin converting enzyme (ACE) is well-known for its role in blood pressure regulation via the renin-angiotensin aldosterone system (RAAS) but also functions in fertility, immunity, haematopoiesis and diseases such as obesity, fibrosis and Alzheimer's dementia. Like ACE, the human homologue ACE2 is also involved in blood pressure regulation and cleaves a range of substrates involved in different physiological processes. Importantly, it is the functional receptor for severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 responsible for the 2020, coronavirus infectious disease 2019 (COVID-19) pandemic. Understanding the interaction between SARS-CoV-2 and ACE2 is crucial for the design of therapies to combat this disease. This review provides a comparative analysis of methodologies and findings to describe how structural biology techniques like X-ray crystallography and cryo-electron microscopy have enabled remarkable discoveries into the structure-function relationship of ACE and ACE2. This, in turn, has enabled the development of ACE inhibitors for the treatment of cardiovascular disease and candidate therapies for the treatment of COVID-19. However, despite these advances the function of ACE homologues in non-human organisms is not yet fully understood. ACE homologues have been discovered in the tissues, body fluids and venom of species from diverse lineages and are known to have important functions in fertility, envenoming and insect-host defence mechanisms. We, therefore, further highlight the need for structural insight into insect and venom ACE homologues for the potential development of novel anti-venoms and insecticides.

High-resolution crystal structures of the botulinum neurotoxin binding domains from subtypes A5 and A6.

Clostridium botulinum neurotoxins (BoNTs) cause flaccid paralysis through inhibition of acetylcholine release from motor neurons; however, at tiny doses, this property is exploited for use as a therapeutic. Each member of the BoNT family of proteins consists of three distinct domains: a binding domain that targets neuronal cell membranes (HC ), a translocation domain (HN ) and a catalytic domain (LC). Here, we present high-resolution crystal structures of the binding domains of BoNT subtypes/A5 (HC /A5) and/A6 (HC /A6). These structures show that the core fold identified in other subtypes is maintained, but with subtle differences at the expected receptor-binding sites.

Molecular Basis for Omapatrilat and Sampatrilat Binding to Neprilysin-Implications for Dual Inhibitor Design with Angiotensin-Converting Enzyme.

Neprilysin (NEP) and angiotensin-converting enzyme (ACE) are two key zinc-dependent metallopeptidases in the natriuretic peptide and kinin systems and renin-angiotensin-aldosterone system, respectively. They play an important role in blood pressure regulation and reducing the risk of heart failure. Vasopeptidase inhibitors omapatrilat and sampatrilat possess dual activity against these enzymes by blocking the ACE-dependent conversion of angiotensin I to the potent vasoconstrictor angiotensin II while simultaneously halting the NEP-dependent degradation of vasodilator atrial natriuretic peptide. Here, we report crystal structures of omapatrilat, sampatrilat, and sampatrilat-ASP (a sampatrilat analogue) in complex with NEP at 1.75, 2.65, and 2.6 Å, respectively. A detailed analysis of these structures and the corresponding structures of ACE with these inhibitors has provided the molecular basis of dual inhibitor recognition involving the catalytic site in both enzymes. This new information will be very useful in the design of safer and more selective vasopeptidase inhibitors of NEP and ACE for effective treatment in hypertension and heart failure.

ACE-domain selectivity extends beyond direct interacting residues at the active site.

Angiotensin-converting enzyme (ACE) is best known for its formation of the vasopressor angiotensin II that controls blood pressure but is also involved in other physiological functions through the hydrolysis of a variety of peptide substrates. The enzyme contains two catalytic domains (nACE and cACE) that have different affinities for ACE substrates and inhibitors. We investigated whether nACE inhibitor backbones contain a unique property which allows them to take advantage of the hinging of nACE. Kinetic analysis showed that mutation of unique nACE residues, in both the S2 pocket and around the prime subsites (S') to their C-domain counterparts, each resulted in a decrease in the affinity of nACE specific inhibitors (SG6, 33RE and ketoACE-13) but it required the combined S2_S' mutant to abrogate nACE-selectivity. However, this was not observed with the non-domain-selective inhibitors enalaprilat and omapatrilat. High-resolution structures were determined for the minimally glycosylated nACE with the combined S2_S' mutations in complex with the ACE inhibitors 33RE (1.8 Å), omapatrilat (1.8 Å) and SG6 (1.7 Å). These confirmed that the affinities of the nACE-selective SG6, 33RE and ketoACE-13 are not only affected by direct interactions with the immediate environment of the binding site, but also by more distal residues. This study provides evidence for a more general mechanism of ACE inhibition involving synergistic effects of not only the S2, S1' and S2' subsites, but also residues involved in the sub-domain interface that effect the unique ways in which the two domains stabilize active site loops to favour inhibitor binding.

Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis.

BACKGROUND: The human eosinophil Charcot-Leyden crystal (CLC) protein is a member of the Galectin superfamily and is also known as galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals that are considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. OBJECTIVE: We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. METHODS: Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by coimmunoprecipitation and coaffinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by using enzyme activity assays, confocal microscopy, and short hairpin RNA knockout of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils. RESULTS: CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases (RNases), namely, eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3), and with murine eosinophil-associated RNases. The interaction is independent of glycosylation and is not inhibitory toward endoRNase activity. Activation of eosinophils with INF-γ induces the rapid colocalization of CLC/Gal-10 with eosinophil-derived neurotoxin/RNS2 and CD63. Short hairpin RNA knockdown of CLC/Gal-10 in human cord blood-derived CD34+ progenitor cells impairs eosinophil granulogenesis. CONCLUSIONS: CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic RNases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation.