Engineering Agatoxin, a Cystine-Knot Peptide from Spider Venom, as a Molecular Probe for In Vivo Tumor Imaging

Background: Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor. Methodology/Principal Findings: We used a rational loop-grafting approach to engineer AgTx variants that bound to α β integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants. Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI. Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins. Conclusions/Significance: In this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging

The role of spermatozoa-zona pellucida interaction in selecting fertilization-competent spermatozoa in humans

Human fertilization begins when a capacitated spermatozoon binds to the zona pellucida (ZP) surrounding a mature oocyte. Defective spermatozoa-ZP interaction contributes to male infertility and is a leading cause of reduced fertilization rates in assisted reproduction treatments (ARTs). Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. This observation suggests that human ZP selectively interacts with competitively superior spermatozoa characterized by high fertilizing capability and genetic integrity. However, direct evidence for ZP-mediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilization-competent spermatozoa in humans. ZP-bound and unbound spermatozoa were respectively collected by a spermatozoa-ZP coincubation assay. The time-course data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZP-binding ability. Immunofluorescent staining indicated that the ZP-bound spermatozoa had significantly higher expression levels of HSPA2 and SPACA3 than the unbound spermatozoa. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound spermatozoa. The results validated the possibility of applying spermatozoa-ZP interaction to select fertilization-competent spermatozoa in ART. This highly selective interaction might also provide diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis

Identification of microbial community in the urban environment: The concordance between conventional culture and nanopore 16S rRNA sequencing

IntroductionMicrobes in the built environment have been implicated as a source of infectious diseases. Bacterial culture is the standard method for assessing the risk of exposure to pathogens in urban environments, but this method only accounts for <1% of the diversity of bacteria. Recently, full-length 16S rRNA gene analysis using nanopore sequencing has been applied for microbial evaluations, resulting in a rise in the development of long-read taxonomic tools for species-level classification. Regarding their comparative performance, there is, however, a lack of information.MethodsHere, we aim to analyze the concordance of the microbial community in the urban environment inferred by multiple taxonomic classifiers, including ARGpore2, Emu, Kraken2/Bracken and NanoCLUST, using our 16S-nanopore dataset generated by MegaBLAST, as well as assess their abilities to identify culturable species based on the conventional culture results.ResultsAccording to our results, NanoCLUST was preferred for 16S microbial profiling because it had a high concordance of dominant species and a similar microbial profile to MegaBLAST, whereas Kraken2/Bracken, which had similar clustering results as NanoCLUST, was also desirable. Second, for culturable species identification, Emu with the highest accuracy (81.2%) and F1 score (29%) for the detection of culturable species was suggested.DiscussionIn addition to generating datasets in complex communities for future benchmarking studies, our comprehensive evaluation of the taxonomic classifiers offers recommendations for ongoing microbial community research, particularly for complex communities using nanopore 16S rRNA sequencing

Engineering Agatoxin, a Cystine-Knot Peptide from Spider Venom, as a Molecular Probe for In Vivo Tumor Imaging

<div><p>Background</p><p>Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the <i>Ecballium elaterium</i> trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor.</p> <p>Methodology/Principal Findings</p><p>We used a rational loop-grafting approach to engineer AgTx variants that bound to α_vβ₃ integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants.</p> <p>Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI.</p> <p>Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins.</p> <p>Conclusions/Significance</p><p>In this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.</p> </div

AgTx, AgRP, and EETI knottins engineered to bind tumor-associated integrins.

<p>(A) Native knottin structures. AgTx IVB (PDB 1OMB), truncated AgRP (PDB 1MR0), and EETI-II (PDB 2ETI), with disulfide bonds shown in gold, and native loops that were mutated to bind tumor-associated integrins shown in red. Structures were rendered in PyMOL. (B) Schematic of protein engineering strategy and sequences of native and engineered knottins used in this study. Conserved cysteine residues are shown in gold, and bars indicate disulfide bond connectivity. The N- and C-termini of AgTx were truncated and the sequences of isoforms IVA and IVB were combined to create a knottin scaffold with no lysine residues (cyan), allowing for site-specific conjugation of AF680 at the N-terminal amino group. The integrin-binding loop from AgRP 7C was grafted into the structurally analogous loop of this new scaffold to create AgTx 7C. Mutated loops are underlined and shown in red. * indicates knottins used for in vivo imaging. EETI RDG contains a scrambled sequence that does not bind integrins, and was used as a negative control.</p

Engineered AgTx 7C variants bind to K562-α_vβ₃ cells with similar IC₅₀ values.

<p>Varying concentrations of AgRP 7C and AgTx 7C variants were incubated with FLAG-AgRP 7A and allowed to compete for binding to integrin receptors expressed on the surface of K562-α_vβ₃ cells. Representative competition binding curves are shown, and are plotted as knottin concentration versus the fraction of FLAG-AgRP 7A bound. IC₅₀ values reported as mean of three experiments ± SD.</p

Unlabeled and AF680-labeled knottins bind U87MG cells with high affinity.

<p>Unlabeled (open squares, dashed line) and AF680-labeled (closed circles, solid line) knottins similarly compete off FLAG-AgRP 7A knottin binding to α_vβ₃ integrins expressed on U87MG glioblastoma cells. Representative competition binding curves shown for (A) AgTx 7C ΔR21, (B) AgRP 7C, (C) EETI 2.5F, and (D) EETI RDG control. IC₅₀ values reported as mean of three experiments ± SD.</p

Relative binding affinities of unlabeled and AF680-labeled knottins on U87MG cells, reported as IC₅₀ values.

<p>(-)  =  no competition observed at highest concentration tested. IC₅₀ values reported as mean of three experiments ± SD.</p