Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection.

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.

MpeV is a lyase isomerase that ligates a doubly linked phycourobilin on the ß-subunit of phycoerythrin I and II in marine Synechococcus.

Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the ß-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into ß-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases.

A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes.

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z' factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.

A bacterial endo-ß-1,4-glucuronan lyase, CUL-I from Brevundimonas sp. SH203, belonging to a novel polysaccharide lyase family.

Cellouronate is a (1,4)-ß-D-glucuronan prepared by TEMPO-mediated oxidation from regenerated cellulose. We have previously isolated a cellouronate-degrading bacterial strain, Brevundimonas sp. SH203, that produces a cellouronate lyase (ß-1,4-glucuronan lyase, CUL-I). In this study, the gene encoding CUL-I was cloned, and the recombinant enzyme was heterologously expressed in Escherichia coli. The predicted CUL-I protein is composed of 426 amino acid residues and includes a putative 21-amino acid signal peptide. The recombinant CUL-I specifically depolymerized ß-1,4-glycoside linkages of cellouronate, and its mode of action was endo-type, like the native CUL-I. Sequence analysis showed CUL-I has no similarity to previously known polysaccharide lyases (PLs), indicating that CUL-I should be classified into a novel PL family.

Cloning and expression of ATP N-glycosidase from the freshwater sponge Ephydatia muelleri.

Previously we had discovered unusual enzymatic activity in the marine sponge Axinella polypoides, ATP N-glycosidase (Reintamm et al., 2003). We show here that the Ephydatia muelleri mRNA encoding protein with PNP_UDP_1 (phosphorylase superfamily) signature is the secreted ATP N-glycosidase. The functionality of the protein was established by recombinant expression in Pichia pastoris. In addition to the enzymatic domain, the full-length protein contains the N-terminal cysteine-rich domain belonging to the subfamily SCP_HrTT-1 (cd05559) of the SCP (sperm coating protein) superfamily (cl00133).

Bacterial tannases: classification and biochemical properties.

Tannin acyl hydrolases, also known as tannases, are a group of enzymes critical for the transformation of tannins. The study of these enzymes, which initially evolved in different organisms to detoxify and/or use these plant metabolites, has nowadays become relevant in microbial enzymology research due to their relevant role in food tannin transformation. Microorganisms, particularly bacteria, are major sources of tannase. Cloning and heterologous expression of bacterial tannase genes and structural studies have been performed in the last few years. However, a systematic compilation of the information related to all recombinant tannases, their classification, and characteristics is missing. In this review, we explore the diversity of heterologously produced bacterial tannases, describing their substrate specificity and biochemical characterization. Moreover, a new classification based on sequence similarity analysis is proposed. Finally, putative tannases have been identified in silico for each group of tannases taking advantage of the use of the "tannase" distinctive features previously proposed.

The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/ß-hydrolase superfamily.

Strain SYK-6 of the bacterium Sphingobium sp. catabolizes lignin-derived biphenyl via a meta-cleavage pathway. In this pathway, LigY is proposed to catalyze the hydrolysis of the meta-cleavage product (MCP) 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate. Here, we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate as the products and determined the kcat and kcat/Km values as 9.3 ± 0.6 s-1 and 2.5 ± 0.2 × 107 m-1 s-1, respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily, unlike previously characterized MCP hydrolases, which are serine-dependent enzymes of the α/ß-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-ß-carboxymuconic-ϵ-semialdehyde decarboxylase, a class III amidohydrolase, with a single zinc ion coordinated by His-6, His-8, His-179, and Glu-282. Interestingly, we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover, substitution of His-223, a conserved residue proposed to activate water in other amidohydrolases, reduced the kcat to a much lesser extent than what has been reported for other amidohydrolases, suggesting that His-223 has a different role in LigY. Substitution of Arg-72, Tyr-190, Arg-234, or Glu-282 reduced LigY activity over 100-fold. On the basis of these results, we propose a catalytic mechanism involving substrate tautomerization, substrate-assisted activation of water for hydrolysis, and formation of a gem-diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C-C-hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.

Dissection of membrane-binding and -remodeling regions in two classes of bacterial phospholipid N-methyltransferases.

Bacterial phospholipid N-methyltransferases (Pmts) catalyze the formation of phosphatidylcholine (PC) via successive N-methylation of phosphatidylethanolamine (PE). They are classified into Sinorhizobium-type and Rhodobacter-type enzymes. The Sinorhizobium-type PmtA protein from the plant pathogen Agrobacterium tumefaciens is recruited to anionic lipids in the cytoplasmic membrane via two amphipathic helices called αA and αF. Besides its enzymatic activity, PmtA is able to remodel membranes mediated by the αA domain. According to the Heliquest program, αA- and αF-like amphipathic helices are also present in other Sinorhizobium- and Rhodobacter-type Pmt enzymes suggesting a conserved architecture of α-helical membrane-binding regions in these methyltransferases. As representatives of the two Pmt families, we investigated the membrane binding and remodeling capacity of Bradyrhizobium japonicum PmtA (Sinorhizobium-type) and PmtX1 (Rhodobacter-type), which act cooperatively to produce PC in consecutive methylation steps. We found that the αA regions in both enzymes bind anionic lipids similar to αA of A. tumefaciens PmtA. Membrane binding of PmtX1 αA is enhanced by its substrate monomethyl-PE indicating a substrate-controlled membrane association. The αA regions of all investigated enzymes remodel spherical liposomes into tubular filaments suggesting a conserved membrane-remodeling capacity of bacterial Pmts. Based on these results we propose that the molecular details of membrane-binding and remodeling are conserved among bacterial Pmts.

Exploring Site-Specific N-Glycosylation of HEK293 and Plant-Produced Human IgA Isotypes.

The full potential of recombinant Immunoglobulin A as therapeutic antibody is not fully explored, owing to the fact that structure-function relationships of these extensively glycosylated proteins are not well understood. Here monomeric IgA1, IgA2m(1), and IgA2m(2) variants of the anti-HER2 antibody (IgG1) trastuzumab were expressed in glyco-engineered Nicotiana benthamiana plants and in human HEK293-6E cells. All three IgA isotypes were purified and subjected to biophysical and biochemical characterization. While no differences in assembly, antigen binding, and glycosylation occupancy were observed, both systems vary tremendously in terms of glycan structures and heterogeneity of glycosylation. Mass-spectrometric analysis of site-specific glycosylation revealed that plant-produced IgAs carry mainly complex-type biantennary N-glycans. HEK293-6E-produced IgAs, on the contrary, showed very heterogeneous N-glycans with high levels of sialylation, core-fucose, and the presence of branched structures. The site-specific analysis revealed major differences between the individual N-glycosylation sites of each IgA subtype. Moreover, the proline-rich hinge region from HEK293-6E cell-derived IgA1 was occupied with mucin-type O-glycans, whereas IgA1 from N. benthamiana displayed numerous plant-specific modifications. Interestingly, a shift in unfolding of the CH2 domain of plant-produced IgA toward lower temperatures can be observed with differential scanning calorimetry, suggesting that distinct glycoforms affect the thermal stability of IgAs.

Identification of immunogenic proteins and evaluation of four recombinant proteins as potential vaccine antigens from Vibrio anguillarum in flounder (Paralichthys olivaceus).

Vibrio anguillarum is a severe bacterial pathogen that can infect a wide range of fish species. Identification of immunogenic proteins and development of vaccine are essential for disease prevention. In this study, immunogenic proteins were screened and identified from V. anguillarum, and then protective efficacy of the immunogenic proteins was evaluated. Immunogenic proteins in V. anguillarum whole cell were detected by Western blotting (WB) using immunized flounder (Paralichthys olivaceus) serum, and then identified by Mass spectrometry (MS). The recombinant proteins of four identified immunogenic proteins were produced and immunized to fish, and then percentages of surface membrane immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBL), total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were measured, respectively. The results showed that five immunogenic proteins, VAA, Groel, OmpU, PteF and SpK, were identified; their recombinant proteins, rOmpU, rGroel, rSpK and rVAA, could induce the proliferation of sIg+ cells in PBL and production of total antibodies, antibodies against V. anguillarum and antibodies against the recombinant proteins; their protection against V. anguillarum showed 64.86%, 72.97%, 21.62% and 78.38% RPS, respectively. The results revealed that the immunoproteomic technique using fish anti-V. anguillarum serum provided an efficient way to screen the immunogenic protein for vaccine antigen. Moreover, the rVAA, rGroel and rOmpU had potential to be vaccine candidates against V. anguillarum infection.

Mycobacteria Encode Active and Inactive Classes of TesB Fatty-Acyl CoA Thioesterases Revealed through Structural and Functional Analysis.

Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.

Identification and Characterization of the Lysine-Rich Matrix Protein Family in Pinctada fucata: Indicative of Roles in Shell Formation.

Mantle can secret matrix proteins playing key roles in regulating the process of shell formation. The genes encoding lysine-rich matrix proteins (KRMPs) are one of the most highly expressed matrix genes in pearl oysters. However, the expression pattern of KRMPs is limited and the functions of them still remain unknown. In this study, we isolated and identified six new members of lysine-rich matrix proteins, rich in lysine, glycine and tyrosine, and all of them are basic matrix proteins. Combined with four members of the KRMPs previously reported, all these proteins can be divided into three subclasses according to the results of phylogenetic analyses: KRMP1-3 belong to subclass KPI, KRMP4-5 belong to KPII, and KRMP6-10 belong to KPIII. Three subcategories of lysine-rich matrix proteins are highly expressed in the D-phase, the larvae and adult mantle. Lysine-rich matrix proteins are involved in the shell repairing process and associated with the formation of the shell and pearl. What's more, they can cause abnormal shell growth after RNA interference. In detail, KPI subgroup was critical for the beginning formation of the prismatic layer; both KPII and KPIII subgroups participated in the formation of prismatic layer and nacreous layer. Compared with different temperatures and salinity stimulation treatments, the influence of changes in pH on KRMPs gene expression was the greatest. Recombinant KRMP7 significantly inhibited CaCO3 precipitation, changed the morphology of calcite, and inhibited the growth of aragonite in vitro. Our results are beneficial to understand the functions of the KRMP genes during shell formation.

Molecular cloning, recombinant expression, and antifungal functional characterization of the lipid transfer protein from Panax ginseng.

OBJECTIVES: To establish an efficient expression system for a fusion protein GST-pgLTP (Lipid Transfer Protein) and to test its antifungal activity. RESULTS: The nucleotide sequence of LTP gene was obtained from Panax ginseng using RT-PCR. The ORF of the cDNA is 363 bp, codING for a protein OF 120 amino acids with a calculated MW of 12.09 kDa. The pgLTP gene with a His6-tag at the C-terminus was cloned into the pGEX-6p1 vector to generate a GST-fusion pgLTP protein construct that was expressed in Escherichia coli Rosetta. Following purification by Ni-NTA, the fusion protein exhibited antifungal activity against five fungi found in ginseng. CONCLUSION: The fusion protein GST-pgLTP has activity against a broad spectrum of phytopathogenic fungi, and can potentially be adapted for production to combat fungal diseases that affect P. ginseng.

Exploitation and characterization of three versatile amidase super family members from Delftia tsuruhatensis ZJB-05174.

Amidases can be assigned into two families according to their amino acid sequences. Three amidases (Dt-Amis) were mined and identified from genome of Delftia tsuruhatensis. Homology analysis demonstrated that Dt-Ami 2 and Dt-Ami 6 belonged to amidase signature (AS) family, while Dt-Ami 7 belonged to nitrilase superfamily. AS amidases were shown to hydrolyze a wide spectrum of amides. Kinetic analysis demonstrated that the extension of chain length of aliphatic amides considerably decreased the Km values, and the turnover numbers (kcat) were high with linear aliphatic amides as substrates. Dt-Ami 2 showed maximum activity near a quite alkaline pH (11.0) and exhibited opposite enantioselectivity to Dt-Ami 6. Furthermore, a novel bioprocess for hydrolysis of 1-cyanocyclohexaneacetamide was developed using Dt-Ami 6 as biocatalyst, resulting in >99% conversion within 1.5h at a substrate loading of 100g/L by 0.5g/L of Escherichia coli cells. On the other hand, nitrilase superfamily amidase only hydrolyzed aliphatic amides. The Km values of Dt-Ami 7 were considerably increased with the extension of chain length of aliphatic amides. The characterized enzymes from different families showed distinct biochemical characteristics and catalytic properties, leading to a better understanding of the two super amidase family members.

Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin.

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance.

Classification of Recombinant Biologics in the EU: Divergence Between National Pharmacovigilance Centers.

BACKGROUND AND OBJECTIVE: Biological medicinal products (biologics) are subject to specific pharmacovigilance requirements to ensure that biologics are identifiable by brand name and batch number in adverse drug reaction (ADR) reports. Since Member States collect ADR data at the national level before the data is aggregated at the European Union (EU) level, it is important that an unambiguous understanding of which medicinal products belong to the biological product category exists. This study aimed to identify the level of consistency between Member States regarding the classification of biologics by national authorities responsible for ADR reporting. METHODS: A sample list of recombinant biologics from the European Medicines Agency database of European Public Assessment Reports was created to analyze five Member States (Belgium, the Netherlands, Spain, Sweden, and the UK) according to which products were classified as biologics by each Member State. We calculated the Fleiss kappa value to analyze interrater reliability. RESULTS: A considerable divergence was identified regarding the classification of the 146 recombinant biologics from the sample list: one Member State classified 100 % of the recombinant biologics from the sample list as biologics, whereas the classification rates in the remaining four Member States ranged between 70 and 88 % for products available on the national market. The interrater reliability for 87 products available on the market in all five Member States was considered poor. CONCLUSION: Discrepancies exist between Member States in the classification of biologics; less divergence exists for common well-known biologics. These findings highlight the need to think about the best approaches to translate EU legislation into national practices. Additionally, we recommend a publicly available and frequently updated list of centrally authorized biologics.

Sequence and structure-based prediction of fructosyltransferase activity for functional subclassification of fungal GH32 enzymes.

Sucrolytic enzymes catalyse sucrose hydrolysis or the synthesis of fructooligosaccharides (FOSs), a prebiotic in human and animal nutrition. FOS synthesis capacity differs between sucrolytic enzymes. Amino-acid-sequence-based classification of FOS synthesizing enzymes would greatly facilitate the in silico identification of novel catalysts, as large amounts of sequence data lie untapped. The development of a bioinformatics tool to rapidly distinguish between high-level FOSs synthesizing predominantly sucrose hydrolysing enzymes from fungal genomic data is presented. Sequence comparison of functionally characterized enzymes displaying low- and high-level FOS synthesis revealed conserved motifs unique to each group. New light is shed on the sequence context of active site residues in three previously identified conserved motifs. We characterized two enzymes predicted to possess low- and high-level FOS synthesis activities based on their conserved motif sequences. FOS data for the enzymes confirmed our successful prediction of their FOS synthesis capacity. Structural comparison of enzymes displaying low- and high-level FOS synthesis identified steric hindrance between nystose and a long loop region present only in low-level FOS synthesizers. This loop is proposed to limit the synthesis of FOS species with higher degrees of polymerization, a phenomenon observed among enzymes displaying low-level FOS synthesis. Conserved sequence motifs surrounding catalytic residues and a distant structural determinant were identifiers of FOS synthesis capacity and allow for functional annotation of sucrolytic enzymes directly from amino acid sequence. The tool presented may also be useful to study the structure-function relationships of ß-fructofuranosidases by identifying mutations present in a group of closely related enzymes displaying similar function.

Characterization and expression of MHC class II alpha and II beta genes in mangrove red snapper (Lutjanus argentimaculatus).

The major histocompatibility complex (MHC) class II plays a key role in adaptive immunity by presenting foreign peptides to CD4(+) T cells and by triggering the adaptive immune response. While the structure and function of MHC class II have been well characterized in mammalian, limited research has been done on fishes. In this study, we characterized the gene structure and expression of MHC class II α (Lunar-DAA) and II ß (Lunar-DAB) of mangrove red snapper (Lutjanus argentimaculatus). Both genes shared, respectively, a high similarity and typical features with other vertebrate MHC class II α and II ß. The phylogenetic analysis of the deduced peptides revealed that both Lunar-DAA and Lunar-DAB were located in the teleost subclass. Western blotting analyses indicated that both MHC class II α and II ß were expressed ubiquitously in immune-related cells, tissues and organs, and that MHC class II α and II ß chains existed mainly as heterodimers. While it was highly expressed in gills, thymus, head kidney (HK), spleen, head kidney macrophage and spleen leucocytes, MHC class II ß chain was expressed with a low abundance in skin, intestine, stomach and heart. The highest expression of MHC class II ß in thymus confirmed the conclusion that thymus is one of the primary lymphoid organs in fishes. The detection of MHC class II αß dimers in HK macrophages and spleen leucocytes indicated that HK macrophages and spleen leucocytes play a critical role in the adaptive immunity in fishes. All these results provide valuable information for understanding the structure of MHC class II α and II ß and their function in immune responses.

Molecular and biochemical characterisation of abomasal nematode parasites Teladorsagia circumcincta and Haemonchus contortus phosphofructokinases and their recognition by the immune host.

Full length cDNAs encoding phosphofructokinase (PFK) were cloned from Teladorsagia circumcincta (TcPFK) and Haemonchus contortus (HcPFK). TcPFK (2361 bp) and HcPFK (2367 bp) cDNA encoded 787 and 789 amino acid proteins respectively. The predicted amino acid sequences showed 98% similarity with each other and 70% with a Caenorhabditis elegans PFK. Substrate binding sites were completely conserved in both proteins. Soluble N-terminal His-tagged PFK proteins were expressed in Escherichia coli strain BL21, purified and characterised. The recombinant TcPFK and HcPFK had very similar kinetic properties: the pH optima were pH 7.0, Km for fructose 6-phosphate was 0.50 ± 0.01 and 0.55 ± 0.01 mM respectively when higher (inhibiting concentration, 0.3 mM) ATP concentration was used and the curve was sigmoidal. The Vmax for TcPFK and HcPFK were 1110 ± 16 and 910 ± 10 nM min(-1 )mg(-1) protein respectively. Lower ATP concentration (non-inhibiting, 0.01 mM) did not change the Vmax for TcPFK and HcPFK (890 ± 10 and 860 ± 12 nM min(-1 )mg(-1) protein) but the substrate affinity doubled and Km for fructose 6-phosphate were 0.20 ± 0.05 and 0.25 ± 0.01 mM respectively. Recognition of TcPFK and HcPFK by mucosal and serum antibodies in nematode exposed animals demonstrates antigenicity and suggests involvement in the host response to nematode infection.

The solution structure of the MANEC-type domain from hepatocyte growth factor activator inhibitor-1 reveals an unexpected PAN/apple domain-type fold.

A decade ago, motif at N-terminus with eight-cysteines (MANEC) was defined as a new protein domain family. This domain is found exclusively at the N-terminus of >400 multi-domain type-1 transmembrane proteins from animals. Despite the large number of MANEC-containing proteins, only one has been characterized at the protein level: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein, as knockout mice die in utero due to placental defects. HAI-1 is an inhibitor of matriptase, hepsin and hepatocyte growth factor (HGF) activator, all serine proteases with important roles in epithelial development, cell growth and homoeostasis. Dysregulation of these proteases has been causatively implicated in pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by the lack of structural information on MANEC. Although many MANEC sequences exist, sequence-based database searches fail to predict structural homology. In the present paper, we present the NMR solution structure of the MANEC domain from HAI-1, the first three-dimensional (3D) structure from the MANEC domain family. Unexpectedly, MANEC is a new subclass of the PAN/apple domain family, with its own unifying features, such as two additional disulfide bonds, two extended loop regions and additional α-helical elements. As shown for other PAN/apple domain-containing proteins, we propose a similar active role of the MANEC domain in intramolecular and intermolecular interactions. The structure provides a tool for the further elucidation of HAI-1 function as well as a reference for the study of other MANEC-containing proteins.