The Evolution and Application of a Novel DNA Aptamer Targeting Bone Morphogenetic Protein 2 for Bone Regeneration.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration.

A combinatorial strategy for HRV 3C protease engineering to achieve the N-terminal free cleavage.

Human rhinovirus 3C protease (HRV 3CP) has a high specificity against the substrate of LEVLFQ↓G at P1' site, which plays an important role in biotechnology and academia as a fusion tag removal tool. However, a non-ignorable limitation is that an extra residue of Gly would remain at the N terminus of the recombinant target protein after cleavage with HRV 3CP, thus potentially causing protein mis-functionality or immunogenicity. Here, we developed a combinatorial strategy by integrating structure-guided library design and high-throughput screening of eYESS approach for HRV 3CP engineering to expand its P1' specificity. Finally, a C3 variant was obtained, exhibiting a broad substrate P1' specificity to recognize 20 different amino acids with the highest activity against LEVLFQ↓M (kcat/KM = 3.72 ± 0.04 mM-1∙s-1). Further biochemical and NGS-mediated substrate profiling analysis showed that C3 variant still kept its substrate stringency at P1 site and good residue tolerance at P2' site, but with an expanded P1' specificity. Structural simulation of C3 indicated a reconstructed S1' binding pocket as well as new interactions with the substrates. Overall, our studies here prompt not only the practical applications and understanding of substrate recognition mechanisms of HRV 3CP, also provide new tools for other enzyme engineering.

A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.

Quantitative Shotgun Proteomic Analysis of Bacteria after Overexpression of Recombinant Spider Miniature Spidroin, MaSp1.

Spider silk has extraordinary mechanical properties, displaying high tensile strength, elasticity, and toughness. Given the high performance of natural fibers, one of the long-term goals of the silk community is to manufacture large-scale synthetic spider silk. This process requires vast quantities of recombinant proteins for wet-spinning applications. Attempts to synthesize large amounts of native size recombinant spidroins in diverse cell types have been unsuccessful. In these studies, we design and express recombinant miniature black widow MaSp1 spidroins in bacteria that incorporate the N-terminal and C-terminal domain (NTD and CTD), along with varying numbers of codon-optimized internal block repeats. Following spidroin overexpression, we perform quantitative analysis of the bacterial proteome to identify proteins associated with spidroin synthesis. Liquid chromatography with tandem mass spectrometry (LC MS/MS) reveals a list of molecular targets that are differentially expressed after enforced mini-spidroin production. This list included proteins involved in energy management, proteostasis, translation, cell wall biosynthesis, and oxidative stress. Taken together, the purpose of this study was to identify genes within the genome of Escherichia coli for molecular targeting to overcome bottlenecks that throttle spidroin overexpression in microorganisms.

Scalable dual column cation exchange affinity chromatography based platform process for recombinant protein purification.

A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting as an initial affinity tag. Combining the heparin-binding tag (HB-tag) with the self-removing iCapTag™ provides a means for removing both tags in a subsequent self-cleaving step. The result is a convenient platform for the purification of diverse tagless proteins with a range of isoelectric points and molecular weights. In this work, we demonstrate a dual column process in which the tagged protein of interest is first captured from an E. coli cell lysate using a cation exchange column via a fused heparin-binding affinity tag. The partially purified protein is then diluted and loaded onto an iCapTag™ split-intein column, washed, and then incubated overnight to release the tagless target protein from the bound tag. Case studies are provided for enhanced green fluorescent protein (eGFP), beta galactosidase (ßgal), maltose binding protein (MBP) and beta lactamase (ßlac), where overall purity and host cell DNA clearance is provided. Overall, the proposed dual column process is shown to be a scalable platform technology capable of accessing both the high dynamic binding capacity of ion exchange resins and the high selectivity of affinity tags for the purification of recombinant proteins.

Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen.

Monoclonal antibodies are one of the fastest growing class of drugs. Nevertheless, relatively few biologics target multispanning membrane proteins because of technical challenges. To target relatively small extracellular regions of multiple membrane-spanning proteins, synthetic peptides, which are composed of amino acids corresponding to an extracellular region of a membrane protein, are often utilized in antibody discovery. However, antibodies to these peptides often do not recognize parental membrane proteins. In this study, we designed fusion proteins in which an extracellular helix of the membrane protein glucose transporter 1 (Glut1) was grafted onto the scaffold protein Adhiron. In the initial design, the grafted fragment did not form a helical conformation. Molecular dynamics simulations of full-length Glut1 suggested the importance of intramolecular interactions formed by surrounding residues in the formation of the helical conformation. A fusion protein designed to maintain such intramolecular interactions did form the desired helical conformation in the grafted region. We then immunized an alpaca with the designed fusion protein and obtained VHH (variable region of heavy-chain antibodies) using the phage display method. The binding of these VHH antibodies to the recombinant Glut1 protein was evaluated by surface plasmon resonance, and their binding to Glut1 on the cell membrane was further validated by flow cytometry. Furthermore, we also succeeded in the generation of a VHH against another integral membrane protein, glucose transporter 4 (Glut4) with the same strategy. These illustrates that our combined biochemical and computational approach can be applied to designing other novel fusion proteins for generating site-specific antibodies.

Structure-function relationship study for sulfated protein therapeutics using hydrophobic interaction chromatography and mass spectrometry.

Protein tyrosine sulfation is a post-translational modification (PTM) that is rarely reported in recombinant therapeutic proteins. However, when sulfation does occur, the additional negative charge from the modification can influence intermolecular interactions and antigen-binding activity, making it a critical quality attribute that necessitates stringent control. In this study, we developed a unique hydrophobic interaction chromatography (HIC) method for the separation and quantification of a therapeutic bispecific antibody with varying degrees of sulfation. Despite the increased surface hydrophilicity of sulfated species, the HIC method provides enhanced retention. Baseline resolution was attained based on the degree of sulfation, independent of other PTMs such as C-terminal amidation and forced deamidation. Further structure-function relationship studies of enriched sulfated bispecific antibody species were conducted using mass spectrometry and fluorescence-linked immunosorbent assay (FLISA). These studies revealed that the tyrosine sulfation modification, which occurs in the complementarity-determining region (CDR), is a critical quality attribute and can adversely impact the antibody's binding to its cognate antigen. The evaluation of sulfation assay using HIC method confirmed it is an effective means for controlling this critical quality attribute.

Recombinant protein production and functional analysis of a M60-like-2 metallopeptidase enzyme from the carcinogenic liver fluke Opisthorchis viverrini.

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov-M60-like-1 and Ov-M60-like-2. Ov-M60-like-1 was previously characterized. Here, we study the Ov-M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini-infected hamsters. Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov-M60-like-2 possess a mucinase activity, together with Ov-M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct.

Recombinant production and characterization of a metal ion-independent Lysophospholipase from a hyperthermophilic archaeon Pyrococcus abyssi DSM25543.

Genome sequence of Pyrococcus abyssi DSM25543 contains a coding sequence (PAB_RS01410) for α/ß hydrolase (WP_010867387.1). Structural analysis revealed the presence of a consensus motif GXSXG and a highly conserved catalytic triad in the amino acid sequence of α/ß hydrolase that were characteristic features of lysophospholipases. A putative lysophospholipase from P. abyssi with its potential applications in oil degumming and starch processing was heterologously produced in E. coli Rosetta (DE3) pLysS in soluble form followed by its purification and characterization. The recombinant enzyme was found to be active at temperature of 40-90 °C and pH 5.5-7.0. However, the enzyme exhibited its optimum activity at 65 °C and pH 6.5. None of the metal ions (Mn2+, Mg2+, Ni2+, Cu2+, Fe2+, Co2+, Zn2+ and Ca2+) being tested had stimulatory effect on lysophospholipase activity. Km and Vmax for hydrolysis of 4-nitrophenyl butyrate were calculated to be 1 ± 0.089 mM and 1637 ± 24.434 U/mg, respectively. It is the first report on the soluble production and characterization of recombinant lysophospholipase from P. abyssi which exhibits its lipolytic activity in the absence of divalent metal ions. Broad substrate specificity, activity and stability at elevated temperatures make recombinant lysophospholipase an ideal candidate for potential industrial applications.

Binding characteristics of pheromone-binding protein 1 in Glyphodes pyloalis to organophosphorus insecticides: Insights from computational and experimental approaches.

Glyphodes pyloalis (Lepidoptera: Pyralidae) is one of the major pests in mulberry production in China, which has developed resistance to various insecticides. Chemoreception is one of the most crucial physiological tactics in insects, playing a pivotal role in recognizing chemical stimuli in the environment, including noxious stimuli such as insecticides. Herein, we obtained recombinant pheromone-binding protein 1 (GpylPBP1) that exhibited antennae-biased expression in G. pyloalis. Ligand-binding assays indicated that GpylPBP1 had the binding affinities to two organophosphorus insecticides, with a higher binding affinity to chlorpyrifos than to phoxim. Computational simulations showed that a mass of nonpolar amino acid residues formed the binding pocket of GpylPBP1 and contributed to the hydrophobic interactions in the bindings of GpylPBP1 to both insecticides. Furthermore, the binding affinities of three GpylPBP1 mutants (F12A, I52A, and F118A) to both insecticides were all significantly reduced compared to those of the GpylPBP1-wild type, suggesting that Phe12, Ile52, and Phe118 residues were crucial binding sites and played crucial roles in the bindings of GpylPBP1 to both insecticides. Our findings can be instrumental in elucidating the effects of insecticides on olfactory recognition in moths and facilitating the development of novel pest management strategies using PBPs as targets based on insect olfaction.

Purification and characterization of an α-l-arabinofuranosidase, α-l-AFase, for hydrolyzed ginsenoside Rc from Bacillus subtilis.

Natural ginsenoside needs to be converted into rare ginsenoside before it can be readily absorbed into the bloodstream for action. In this study, an α-l-arabinofuranosidase (α-l-AFase) gene Bsafs2 was cloned from Bacillus subtilis (B. subtilis). Bsafs2 was ligated to the expression vector pET28a(+), and the expression vector was constructed and transformed into Escherichia coli (E. coli) BL21 heterologous recombinant expression to obtain α-l-AFase. α-l-AFase can hydrolyze at the C20 site of Ginsenoside Rc to obtain rare ginsenoside Rd. Studies on the enzymatic property showed that α-l-AFase had good tolerance to ethanol, glucose, and l-arabinose. The optimum temperature of α-l-AFase was 40 °C and pH = 5.5. Kinetic parameters Km of α-l-AFase for pNPαAraf and Ginsenoside Rc were 1.93 and 8.9 mmol/L, the Vmax were 26 and 154 µmol/min/mg, the Kcat were 24.14 and 1.48 S-1, respectively. This study provides the enzyme source for the biotransformation of Ginsenoside Rc.

Characterization of a novel recombinant D-mannose isomerase from Bifidobacterium bifidum and its catalytic mechanism.

Due to the increasing demand for health-conscious and environmentally friendly products, D-mannose has gained significant attention as a natural, low-calorie sweetener. The use of D-mannose isomerases (D-MIases) for D-mannose production has emerged as a prominent area of research, offering superior advantages compared with conventional methods such as plant extraction and chemical synthesis. In this study, a gene encoding D-MIase was cloned from Bifidobacterium and expressed in E. coli BL21 (DE3). The heterologously expressed enzyme, Bifi-mannose, formed a trimer with a molecular weight of 146.3 kDa and a melting temperature (Tm) of 63.39 ± 1.3 °C. Bifi-mannose exhibited optimal catalytic activity at pH 7.5 and 55 °C, and retained more than 80% of its activity after a 3-hour incubation at 55 °C, demonstrating excellent thermal stability. The Km, Vmax, and kcat/Km values of Bifi-mannose for D-fructose isomerization were determined as 538.7 ± 62.5 mM, 11.7 ± 0.9 µmol·mg1·s1, and 1.02 ± 0.3 mM1·s1, respectively. Notably, under optimized conditions, catalytic yields of 29.4, 87.1, and 148.5 mg·mL1 were achieved when using 100, 300, and 500 mg·mL1 of D-fructose as substrates, resulting in a high conversion rate (29%). Furthermore, kinetic parameters and molecular docking studies revealed that His387 residue primarily participates in the opening of the pyranose ring, while His253 acts as a basic catalyst in the isomerization process.

Structure and biochemical characterization of l-2-hydroxyglutarate dehydrogenase and its role in the pathogenesis of l-2-hydroxyglutaric aciduria.

l-2-hydroxyglutarate dehydrogenase (L2HGDH) is a mitochondrial membrane-associated metabolic enzyme, which catalyzes the oxidation of l-2-hydroxyglutarate (l-2-HG) to 2-oxoglutarate (2-OG). Mutations in human L2HGDH lead to abnormal accumulation of l-2-HG, which causes a neurometabolic disorder named l-2-hydroxyglutaric aciduria (l-2-HGA). Here, we report the crystal structures of Drosophila melanogaster L2HGDH (dmL2HGDH) in FAD-bound form and in complex with FAD and 2-OG and show that dmL2HGDH exhibits high activity and substrate specificity for l-2-HG. dmL2HGDH consists of an FAD-binding domain and a substrate-binding domain, and the active site is located at the interface of the two domains with 2-OG binding to the re-face of the isoalloxazine moiety of FAD. Mutagenesis and activity assay confirmed the functional roles of key residues involved in the substrate binding and catalytic reaction and showed that most of the mutations of dmL2HGDH equivalent to l-2-HGA-associated mutations of human L2HGDH led to complete loss of the activity. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of L2HGDH and provide insights into the functional roles of human L2HGDH mutations in the pathogeneses of l-2-HGA.

Comprehensive evaluation of recombinant lactate dehydrogenase production from inclusion bodies.

A broad application spectrum ranging from clinical diagnostics to biosensors in a variety of sectors, makes the enzyme Lactate dehydrogenase (LDH) highly interesting for recombinant protein production. Expression of recombinant LDH is currently mainly carried out in uncontrolled shake-flask cultivations leading to protein that is mostly produced in its soluble form, however in rather low yields. Inclusion body (IB) processes have gathered a lot of attention due to several benefits like increased space-time yields and high purity of the target product. Thus, to investigate the suitability of this processing strategy for ldhL1 production, a fed-batch fermentation steering the production of IBs rather than soluble product formation was developed. It was shown that the space-time-yield of the fermentation could be increased almost 3-fold by increasing qs to 0.25 g g-1 h-1 which corresponds to 21% of qs,max, and keeping the temperature at 37°C after induction. Solubilization and refolding unit operations were developed to regain full bioactivity of the ldhL1. The systematic approach in screening for solubilization and refolding conditions revealed buffer compositions and processing strategies that ultimately resulted in 50% product recovery in the refolding step, revealing major optimization potential in the downstream processing chain. The recovered ldhL1 showed an optimal activity at pH 5.5 and 30∘C with a high catalytic activity and KM values of 0.46 mM and 0.18 mM for pyruvate and NADH, respectively. These features, show that the here produced LDH is a valuable source for various commercial applications, especially considering low pH-environments.

Ferritin-based fusion protein shows octameric deadlock state of self-assembly.

Ferritin is a universal protein complex responsible for iron perception in almost all living organisms and has applications from fundamental biophysics to drug delivery and structure-based immunogen design. Different platforms based on ferritin share similar technological challenges limiting their development - control of self-assembling processes of ferritin itself as well as ferritin-based chimeric recombinant protein complexes. In our research, we studied self-assembly processes of ferritin-based protein complexes under different expression conditions. We fused a ferritin subunit with a SMT3 protein tag, a homolog of human Small Ubiquitin-like Modifier (SUMO-tag), which was taken to destabilize ferritin 3-fold channel contacts and increase ferritin-SUMO subunits solubility. We first obtained the octameric protein complex of ferritin-SUMO (8xFer-SUMO) and studied its structural organization by small-angle X-ray scattering (SAXS). Obtained SAXS data correspond well with the high-resolution models predicted by AlphaFold and CORAL software of an octameric assembly around the 4-fold channel of ferritin without formation of 3-fold channels. Interestingly, three copies of 8xFer-SUMO do not assemble into 24-meric globules. Thus, we first obtained and structurally characterized ferritin-based self-assembling oligomers in a deadlock state. Deadlock oligomeric states of ferritin extend the known scheme of its self-assembly process, being new potential tools for a number of applications. Finally, our results might open new directions for various biotechnological platforms utilizing ferritin-based tools.

Nanoclay-Modified Hyaluronic Acid Microspheres for Bone Induction by Sustained rhBMP-2 Delivery.

Microspheres (MSs) are ideal candidates as biological scaffolds loading with growth factors or cells for bone tissue engineering to repair irregular alveolar bone defects by minimally invasive injection. However, the high initial burst release of growth factor and low cell attachment limit the application of microspheres. The modification of microspheres often needs expensive experiments facility or complex chemical reactions, which is difficult to achieve and may bring other problems. In this study, a sol-grade nanoclay, laponite XLS is used to modify the surface of MSs to enhance its affinity to either positively or negatively charged proteins and cells without changing the interior structure of the MSs. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is used as a representation of growth factor to check the osteoinduction ability of laponite XLS-modified MSs. By modification, the protein sustained release, cell loading, and osteoinduction ability of MSs are improved. Modified by 1% laponite XLS, the MSs can not only promote osteogenic differentiation of MC3T3-E1 cells by themselves, but also enhance the effect of the rhBMP-2 below the effective dose. Collectively, the study provides an easy and viable method to modify the biological behavior of microspheres for bone tissue regeneration.

High purity of human secreted phospholipase A2 group IIE in Pichia pastoris using basal salts medium comparison with YPD medium.

Secreted phospholipase A2s (sPLA2s) are a group of enzymes with 6-8 disulfide bonds that participate in numerous physiological processes by catalyzing the hydrolysis of phospholipids at the sn-2 position. Due to their high content of disulfide bonds and hydrolytic activity toward cell membranes, obtaining the protein of sPLA2s in the soluble and active form is challenging, which hampers their functional study. In this study, one member of recombinant human sPLA2s, tag-free group IIE (GIIE), was expressed in Pichia pastoris. The protein GIIE was purified from the crude culture supernatant by a two-step chromatography procedure, a combination of cation exchange and size-exclusion chromatography. In the shake flask fermentation, Protein of GIIE with higher purity was successfully obtained, using basal salts medium (BSM) instead of YPD medium. In the large-scale fermentation, each liter of BSM produced a final yield of 1.2 mg pure protein GIIE. This protocol will facilitate further research of GIIE and provide references for the production of other sPLA2 members.

Genome-wide identification and in-silico analysis of papain-family cysteine protease encoding genes in Tetrahymena thermophila.

Tetrahymena thermophila is a promising host for recombinant protein production, but its utilization in biotechnology is mostly limited due to the presence of intracellular and extracellular papain-family cysteine proteases (PFCPs). In this study, we employed bioinformatics approaches to investigate the T. thermophila PFCP genes and their encoded proteases (TtPFCPs), the most prominent protease family in the genome. Results from the multiple sequence alignment, protein modeling, and conserved motif analyses revealed that all TtPFCPs showed considerably high homology with mammalian cysteine cathepsins and contained conserved amino acid motifs. The total of 121 TtPFCP-encoding genes, 14 of which were classified as non-peptidase homologs, were found. Remaining 107 true TtPFCPs were divided into four distinct subgroups depending on their homology with mammalian lysosomal cathepsins: cathepsin L-like (TtCATLs), cathepsin B-like (TtCATBs), cathepsin C-like (TtCATCs), and cathepsin X-like (TtCATXs) PFCPs. The majority of true TtPFCPs (96 out of the total) were in TtCATL-like peptidase subgroup. Both phylogenetic and chromosomal localization analyses of TtPFCPs supported the hypothesis that TtPFCPs likely evolved through tandem gene duplication events and predominantly accumulated on micronuclear chromosome 5. Additionally, more than half of the identified TtPFCP genes are expressed in considerably low quantities compared to the rest of the TtPFCP genes, which are expressed at a higher level. However, their expression patterns fluctuate based on the stage of the life cycle. In conclusion, this study provides the first comprehensive in-silico analysis of TtPFCP genes and encoded proteases. The results would help designing an effective strategy for protease knockout mutant cell lines to discover biological function and to improve the recombinant protein production in T. thermophila.

POFUT1-mediated O-fucosylation of glycoproteins expressed in the baculovirus Sf9 insect cell expression system.

The baculovirus-insect cell expression system allows addition of O-fucose to EGF-like domains of glycoproteins, following the action of the protein O-fucosyltransferase 1 named POFUT1. In this study, recombinant Spodoptera frugiperda POFUT1 from baculovirus-infected Sf9 cells was compared to recombinant Mus musculus POFUT1 produced by CHO cells. Contrary to recombinant murine POFUT1 carrying two hybrid and/or complex type N-glycans, Spodoptera frugiperda POFUT1 exhibited paucimannose N-glycans, at least on its highly evolutionary conserved across Metazoa NRT site. The abilities of both recombinant enzymes to add in vitro O -fucose to EGF-like domains of three different recombinant mammalian glycoproteins were then explored. In vitro POFUT1-mediated O-fucosylation experiments, followed by click chemistry and blot analyses, showed that Spodoptera frugiperda POFUT1 was able to add O-fucose to mouse NOTCH1 EGF-like 26 and WIF1 EGF-like 3 domains, similarly to the murine counterpart. As proved by mass spectrometry, full-length human WNT Inhibitor Factor 1 expressed by Sf9 cells was also modified with O-fucose. However, Spodoptera frugiperda POFUT1 was unable to modify the single EGF-like domain of mouse PAMR1 with O-fucose, contrary to murine POFUT1. Absence of orthologous proteins such as PAMR1 in insects may explain the enzyme's difficulty in adding O-fucose to a domain that it never encounters naturally.

Probing the mechanism by which the retinal G protein transducin activates its biological effector PDE6.

Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein transducin (GαT). Recently, we presented a cryo-EM structure for a complex between two GTP-bound recombinant GαT subunits and native PDE6, that included a bivalent antibody bound to the C-terminal ends of GαT and the inhibitor vardenafil occupying the active sites on the PDEα and PDEß subunits. We proposed GαT-activated PDE6 by inducing a striking reorientation of the PDEγ subunits away from the catalytic sites. However, questions remained including whether in the absence of the antibody GαT binds to PDE6 in a similar manner as observed when the antibody is present, does GαT activate PDE6 by enabling the substrate cGMP to access the catalytic sites, and how does the lipid membrane enhance PDE6 activation? Here, we demonstrate that 2:1 GαT-PDE6 complexes form with either recombinant or retinal GαT in the absence of the GαT antibody. We show that GαT binding is not necessary for cGMP nor competitive inhibitors to access the active sites; instead, occupancy of the substrate binding sites enables GαT to bind and reposition the PDE6γ subunits to promote catalytic activity. Moreover, we demonstrate by reconstituting GαT-stimulated PDE6 activity in lipid bilayer nanodiscs that the membrane-induced enhancement results from an increase in the apparent binding affinity of GαT for PDE6. These findings provide new insights into how the retinal G protein stimulates rapid catalytic turnover by PDE6 required for dim light vision.