Interactions of barley α-amylase isozymes with Ca2 + , substrates and proteinaceous inhibitors

α-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/α)₈-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca^2?+?, different substrates, and proteinaceous inhibitors for α-amylases. Isozyme specific effects of Ca^2?+? on the 80% sequence identical barley α-amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca^2?+? with identical ligands. A fully hydrated fourth Ca^2?+? at the interface of the AMY2/barley α-amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca^2?+? occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca^2?+?. Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered ‘sugar tongs’ site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley α-amylases are reported in this review.     

Dissecting intrinsic and ligand-induced structural communication in the β3 headpiece of integrins

BackgroundGraph theory is widely used to dissect structural communication in biomolecular systems. Here, graph theory-based approaches were applied to the headpiece of integrins, adhesion cell-surface receptors that transmit signals across the plasma membranes.MethodsProtein Structure Network (PSN) analysis incorporating dynamic information either from molecular dynamics simulations or from Elastic Network Models was applied to the β3 domains from integrins α_Vβ₃ and α_IIbβ₃ in their apo and ligand-bound states.ResultsClosed and open states of the β headpiece are characterized by distinct allosteric communication pathways involving highly conserved amino acids at the two different α/β interfaces in the βI domain, the closed state being prompted to the closed-to-open transition. In the closed state, pure antagonism is associated with the establishment of communication pathways that start from the ligand, pass through the β1/α3,α4 interface, and end up in the hybrid domain by involving the Y110-Q82 link, which is weakened in the agonist-bound states.ConclusionsAllosteric communication in integrins relies on highly conserved and functionally relevant amino acid residues. The αβα-sandwich architecture of integrin βI domain dictates the structural communication between ligand binding site and hybrid domain. Differently from agonists, pure antagonists are directly involved in allosteric communication pathways and exert long-distance strengthening of the βI/hybrid interface. Release of the structure network in the ligand binding site is associated with the close-to-open transition accompanying the activation process.General significanceThe study strengthens the power of graph-based analyses to decipher allosteric communication intrinsic to protein folds and modified by functionally different ligands.     

Solution structure and backbone dynamics of the Trypanosoma cruzi cysteine protease inhibitor chagasin

A Trypanosoma cruzi cysteine protease inhibitor, termed chagasin, is the first characterized member of a new family of tight-binding cysteine protease inhibitors identified in several lower eukaryotes and prokaryotes but not present in mammals. In the protozoan parasite T.cruzi, chagasin plays a role in parasite differentiation and in mammalian host cell invasion, due to its ability to modulate the endogenous activity of cruzipain, a lysosomal-like cysteine protease. In the present work, we determined the solution structure of chagasin and studied its backbone dynamics by NMR techniques. Structured as a single immunoglobulin-like domain in solution, chagasin exerts its inhibitory activity on cruzipain through conserved residues placed in three loops in the same side of the structure. One of these three loops, L4, predicted to be of variable length among chagasin homologues, is flexible in solution as determined by measurements of (15)N relaxation. The biological implications of structural homology between chagasin and other members of the immunoglobulin super-family are discussed.     

The X-ray structure of the zinc transporter ZnuA from Salmonella enterica discloses a unique triad of zinc-coordinating histidines

ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the cluster 9 group of ATP-binding cassette-type periplasmic Zn- and Mn-binding proteins. In Gram-negative bacteria, the ZnuABC system is essential for zinc uptake and homeostasis and is an important determinant of bacterial resistance to the host defense mechanisms. The cluster 9 members share a two (α/β)₄ domain architecture with a long α-helix connecting the two domains. In the Zn-specific proteins, the so-called α3c and the α4 helices are separated by an insert of variable length, rich in histidine and negatively charged residues. This distinctive His-rich loop is proposed to play a role in the management of zinc also due to its location at the entrance of the metal binding site located at the domain interface. The known Synechocystis 6803 and Escherichia coli ZnuA structures show the same metal coordination involving three conserved histidines and a glutamic acid or a water molecule as fourth ligand. The structures of Salmonella enterica ZnuA, with a partially or fully occupied zinc binding site, and of a deletion mutant missing a large part of the His-rich loop revealed unexpected differences in the metal-coordinating ligands, as histidine 140 from the mobile (at the C-terminal) part of the loop substitutes the conserved histidine 60. This unforeseen coordination is rendered possible by the “open conformation” of the two domains. The possible structural determinants of these peculiarities and their functional relevance are discussed.     

Evolution of β-amylase: Patterns of variation and conservation in subfamily sequences in relation to parsimony mechanisms

Soybean and sweet potato β-amylases are structured as α/β barrels and the same kind of folding may account for all known β-amylases. We provide a comprehensive analysis of both protein and DNA (coding region) sequences of β-amylases. The aim of the study is to contribute to the knowledge of the evolutionary molecular relationships among all known β-amylases. Our approach combines the identification of the putative eightfold structural core formed by β-strands with a complete multi-alignment analysis of all known sequences. Comparing putative β-amylase (α/β)₈ cores from plants and microorganisms, two differentiated versions of residues at the packing sites, and a unique set of eight identical residues at the C-terminal catalytical site are observed, indicating early evolutionary divergence and absence of localized three-dimensional evolution, respectively. A new analytical approach has been developed in order to work out conserved motifs for β-amylases, mostly related with the enzyme activity. This approach appears useful as a new routine to find sets of motifs (each set being known as a fingerprint) in protein families. We demonstrate that the evolutionary mechanism for β-amylases is a combination of parsimonious divergence at three distinguishable rates in relation to the functional signatures, the barrel scaffold, and α-helix-containing loops. © 1996 Wiley-Liss, Inc.     

Evolutionary Analysis of the ErbB Receptor and Ligand Families

We have compared all available deduced protein sequences of the ErbB family of receptors and their ligands. Analysis of the aligned sequences of the receptors indicates that there are some differences in the receptors that are specific to invertebrates. In addition, comparison of the vertebrate ErbB receptors suggest that a gene duplication event generated two ancestral receptors, the ErbB3/ErbB4 precursor and the ErbB1/ErbB2 precursor. Subsequent gene duplications of these precursors generated the four receptors present in mammals. Analysis of the sequences for the known ligands of the ErbB receptors suggests that the vertebrate ligands segregate into the ErbB1 ligands and the ErbB3/ErbB4 ligands, paralleling the evolution of the receptors; however, it is difficult to ascertain any correlation between the invertebrate and the vertebrate ligands. Even though ErbB3 is kinase-impaired, there is significant conservation of the kinase domain within the vertebrate lineage (human, rat, and F. rubripes), suggesting some function for this domain other than kinase activity, such as mediating protein–protein interactions that are involved in receptor dimerization and/or activation of the kinase domain of the heterodimerization partner. To date, no ligand for ErbB2 has been identified, and comparison of the extracellular domains of ErbB2 reveals two regions that are not conserved across the mammalian species. These two regions of divergence align with sequences in ErbB1 that have been shown to be proximal to the amino-terminus and to the carboxyl-terminal region, respectively, of bound EGF. Further, one of these regions contains an insertion, relative to the other members of the mammalian ErbB family, which might affect the ligand binding site and provide a structural basis for this receptor's apparent inability to bind ligand independently. Received: 8 September 1999 / Accepted: 17 January 2000     

A NPxY-independent beta5 integrin activation signal regulates phagocytosis of apoptotic cells

Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the β chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 (β5) integrin cDNA was expressed in αv positive, β5 and β3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, β5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing β5 mutant (Y750A) abrogated adhesion and β5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of β5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a β5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 μm diameter microspheres developed as apoptotic cell mimetics. The critical sequences in β5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of β5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the β5 cytoplasmic tail for adhesion and phagocytosis.     

Solution structure of HtrA PDZ domain from Streptococcus pneumoniae and its interaction with YYF-COOH containing peptides

High-temperature requirement A (HtrA), a highly conserved family of serine protease, plays crucial roles in protein quality control in prokaryotes and eukaryotes. The HtrA protein contains a C-terminal PDZ domain that mediates the proteolytic activity. Here we reported the solution structure of the HtrA PDZ domain from Streptococcus pneumoniae by NMR spectroscopy. Our results showed that the structure of HtrA PDZ domain, which contains three α-helices and five β-strands, illustrates conservation within the canonical PDZ domains. In addition, we demonstrated the interactions between S. pneumoniae HtrA PDZ domain and peptides with the motif XXX–YYF–COOH by surface plasmon resonance. Besides, we identified the ligand binding surface and the critical residues responsible for ligand binding of HtrA PDZ domain by chemical shift perturbation and site-directed mutagenesis.     

A novel class of cysteine protease receptors that mediate lysosomal transport

The transport of lysosomal proteins is, in general, mediated by mannose 6-phosphate receptors via carbohydrate modifications. Here, we describe a novel class of receptors that regulate the transport of lysosomal hydrolases in the enteric protozoan Entamoeba histolytica, which is a good model organism to investigate membrane traffic. A novel 110 kDa cysteine protease (CP) receptor (CP-binding protein family 1, CPBF1) was initially discovered by affinity co-precipitation of the major CP (EhCP-A5), which plays a pivotal role in the pathogenesis of E. histolytica. We demonstrated that CPBF1 regulates EhCP-A5 transport from the endoplasmic reticulum to lysosomes and its binding to EhCP-A5 is independent of carbohydrate modifications. Repression of CPBF1 by gene silencing led to the accumulation of the unprocessed form of EhCP-A5 in the non-acidic compartment and the mis-secretion of EhCP-A5, suggesting that CPBF1 is involved in the trafficking and processing of EhCP-A5. The CPBF represents a new class of transporters that bind to lysosomal hydrolases in a carbohydrate-independent fashion and regulate their trafficking, processing and activation and, thus, regulate the physiology and pathogenesis of E. histolytica.     

Crystal structure of glycoside hydrolase family 127 β-l-arabinofuranosidase from Bifidobacterium longum

Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-l-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-l-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β-l-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-l-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn²⁺ coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-l-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.     

Contributions of conserved residues at the gating interface of glycine receptors

Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln.     

整联蛋白与其配体结合的分子机制研究进展

整联蛋白是细胞表面的主要膜受体,具有介导细胞与细胞基质间的黏附及病毒吸附细胞等功能。αⅠ结构域型整联蛋白和非αⅠ结构域型整联蛋白与配体结合的方式各异。对整联蛋白-配体复合物分子结构的研究表明,整联蛋白利用其配体结合位点的αMIDAS（依赖金属离子的吸附位点）、βMIDAS与各种类型配体结合。     

Formation of O-Alkylhomoserines from Alcohols,by Yeasts Capable of Assimilating 1,2-Propanediol

The less reactive SH groups of soybean β-amylase, SH4, SH5, and SH6, were modified with p-chloromercuribenzoic acid or N-ethylmaleimide, after the reactive SH groups, SHI, SH2, and SH3, were blocked with 5,5′-dithiobis-(2-nitrobenzoic acid) and cyanide. The enzyme activity decreased, accompanied by the modification of SH4. α-Cyclodextrin protected SH4 from the modification more effectively than maltose. The SH4-modified enzyme still bound to glucose, maltose, and α-cyclodextrin. SH4 was concerned with neither the catalysis nor substrate binding but its large substituent affected the substrate binding site. The sequencing of the 5-(iodoacetoamidoethyl)-aminonaphthalene-1-sulfonate-labeled peptides showed that SH4, SH5, and SH6 are Cys343, Cys82, and Cys208, respectively. Comparison of the primary structure of β-amylases also showed that the sequence around SH4 (Cys343), as well as SH2 (Cys95), is strongly conserved between higher plant and bacterial β-amylases. These results agree with the structure model deduced from X-ray crystallography of soybean β-amylase.     

Characterization of the zinc-binding site of the histidine-proline-rich glycoprotein associated with rabbit skeletal muscle AMP deaminase

The AMP deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn(2+) ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-proline-rich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zinc-binding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP deaminase.     

Elucidating the molecular interaction of Zebrafish (Danio rerio) peptidoglycan recognition protein 2 with diaminopimelic acid and lysine type peptidoglycans using in silico approaches

Abstract

Peptidoglycan recognition proteins (PGRPs) belong to the family of pattern recognition receptor, represent the major constituent of innate immunity. Although PGRPs are structurally conserved through evolution, their involvement in innate immunity is different in vertebrates and invertebrates. They are highly specific towards recognition of ligands and can hydrolyze bacterial peptidoglycans (PGNs). Zebrafish PGRPs (zPGRPs) have both peptidoglycans lytic amidase activity and broad-spectrum bactericidal activity, but far less is known about how these receptors recognize these microbial ligands. Such studies are hindered due to lack of structural and functional configuration of zPGRPs. Therefore, in this study, we predicted the three-dimensional structure of zPGRP2 through theoretical modeling, investigated the conformational and dynamic properties through molecular dynamics simulations. Molecular docking study revealed the microbial ligands, that is, muramyl pentapeptide–DAP , muramyl pentapeptide–LYS, muramyl tripeptide–DAP, muramyl tripeptide–Lys, muramyl tetrapeptide–DAP, muramyl tetrapeptide–LYS and tracheal cytotoxin interacts with the conserved amino acids of the ligand recognition site comprised of β1, α2, α4, β4 and loops connecting β1 ? α2, α2 ? β2, β3 ? β4 and α4 ? α5. Conserved His31, His32, Ala34, Ile35, Pro36, Lys38, Asp60, Trp61, Trp63, Ala89, His90, Asp106, His143 and Arg144 are predicted to essential for binding and provides stability to these zPGRP–PGN complexes. Our study provides basic molecular information for further research on the immune mechanisms of PGRP’s in Zebrafish. The plasticity of the zPGRP’s binding site revealed by these microbial ligands suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges in the future.

Communicated by Ramaswamy H. Sarma     

Design, synthesis, and subtype selectivity of 3,6-disubstituted β-carbolines at Bz/GABA(A)ergic receptors. SAR and studies directed toward agents for treatment of alcohol abuse

A series of 3,6-disubstituted β-carbolines was synthesized and evaluated for their in vitro affinities at α(x)β(3)γ(2) GABA(A)/benzodiazepine receptor subtypes by radioligand binding assays in search of α(1) subtype selective ligands to treat alcohol abuse. Analogues of β-carboline-3-carboxylate-t-butyl ester (βCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted β-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo-βCCt (5). The bivalent ligands of βCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure-activity relationships (SAR) to larger ligands. Based on the pharmacophore/receptor model, a preliminary SAR study on 34 analogues illustrated that large substituents at position-6 of the β-carbolines were well tolerated. As expected, these groups are proposed to project into the extracellular domain (L(Di) region) of GABA(A)/Bz receptors (see 32 and 33). Moreover, substituents located at position-3 of the β-carboline nucleus exhibited a conserved stereo interaction in lipophilic pocket L(1), while N(2) presumably underwent a hydrogen bonding interaction with H(1). Three novel β-carboline ligands (βCCt, 3PBC and WYS8), which preferentially bound to α1 BzR subtypes permitted a comparison of the pharmacological efficacies with a range of classical BzR antagonists (flumazenil, ZK93426) from several different structural groups and indicated these β-carbolines were 'near GABA neutral antagonists'. Based on the SAR, the most potent (in vitro) α(1) selective ligand was the 6-substituted acetylenyl βCCt (WYS8, 7). Earlier both βCCt and 3PBC had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats but had little or no effect on sucrose self-administration.(1-3) Moreover, these two β-carbolines were orally active, and in addition, were anxiolytic in P rats but were only weakly anxiolytic in rodents. These data prompted the synthesis of the β-carbolines presented here.     

Site-directed mutagenesis of beta-adrenergic receptors. Identification of conserved cysteine residues that independently affect ligand binding and receptor activation

Using site-directed mutagenesis of the human beta 2-adrenergic receptor and continuous expression in B-82 cells, the role of 3 conserved cysteines in transmembrane domains and 2 conserved cysteines in the third extracellular domain in receptor function was examined. Cysteine was replaced with serine in each mutant receptor as this amino acid is similar to cysteine in size but it cannot form disulfide linkages. Replacement of cysteine residues 77 and 327, in the second and seventh transmembrane-spanning domains, respectively, had no effect on ligand binding or the ability of the receptor to mediate isoproterenol stimulation of adenylate cyclase. Substitution of cysteine 285, in the sixth transmembrane domain of the receptor, produced a mutant receptor with normal ligand-binding properties but a significantly attenuated ability to mediate stimulation of adenylate cyclase. Mutation of cysteine residues 190 and 191, in the third extracellular loop of the beta 2 receptor, had qualitatively similar effects on ligand binding and isoproterenol-mediated stimulation of adenylate cyclase. Replacement of either of these residues with serine produced mutant receptors that displayed a marked loss in affinity for both beta-adrenergic agonists and antagonists. Replacement of both cysteine 190 and 191 with serine had an even greater effect on the ability of the receptor to bind ligands. Consistent with the loss of Ser190 and/or Ser191 mutant receptor affinity for agonists was a corresponding shift to the right in the dose-response curve for isoproterenol-induced increases in intracellular cyclic AMP concentrations in cells expressing the mutant receptors. These data implicate one of the conserved transmembrane cysteine residues in the human beta 2-adrenergic receptor in receptor activation by agonists and also suggest that conserved cysteine residues in an extracellular domain of the receptor may be involved in ligand binding.     

The Cysteine Residue in the Carboxyl-Terminal Domain of the m2 Muscarinic Acetylcholine Receptor Is Not Required for Receptor-Mediated Inhibition of Adenylate Cyclase

Muscarinic acetylcholine receptors (mAChRs) share with many other receptors of the guanine nucleotide-binding protein-coupled receptor family a highly conserved cysteine residue in the putative cytoplasmic carboxyl-terminal region of the protein. Because elimination of this cysteine in the beta 2-adrenergic receptor has been reported to decrease functional responsiveness, we determined if this cysteine residue is essential for mAChR-effector coupling by replacing Cys457 of the m2 mAChR with glycine and expressing wild-type and mutant receptor in Chinese hamster ovary (CHO) cells. The mutant and wild-type receptors exhibited similar affinities for binding of muscarinic ligands. In addition, the mutation did not affect cell surface localization or receptor-mediated inhibition of adenylate cyclase. These results indicate that the cysteine residue in the carboxyl-terminal domain of the m2 mAChR is not required for ligand binding or mAChR-mediated inhibition of adenylate cyclase in CHO cells.     

Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and ��-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.