Enhanced mapping of small-molecule binding sites in cells.

Photoaffinity probes are routinely utilized to identify proteins that interact with small molecules. However, despite this common usage, resolving the specific sites of these interactions remains a challenge. Here we developed a chemoproteomic workflow to determine precise protein binding sites of photoaffinity probes in cells. Deconvolution of features unique to probe-modified peptides, such as their tendency to produce chimeric spectra, facilitated the development of predictive models to confidently determine labeled sites. This yielded an expansive map of small-molecule binding sites on endogenous proteins and enabled the integration with multiplexed quantitation, increasing the throughput and dimensionality of experiments. Finally, using structural information, we characterized diverse binding sites across the proteome, providing direct evidence of their tractability to small molecules. Together, our findings reveal new knowledge for the analysis of photoaffinity probes and provide a robust method for high-resolution mapping of reversible small-molecule interactions en masse in native systems.

Chemoproteomic development of SLC15A4 inhibitors with anti-inflammatory activity.

SLC15A4 is an endolysosome-resident transporter linked with autoinflammation and autoimmunity. Specifically, SLC15A4 is critical for Toll-like receptors (TLRs) 7-9 as well as nucleotide-binding oligomerization domain-containing protein (NOD) signaling in several immune cell subsets. Notably, SLC15A4 is essential for the development of systemic lupus erythematosus in murine models and is associated with autoimmune conditions in humans. Despite its therapeutic potential, the availability of quality chemical probes targeting SLC15A4 functions is limited. In this study, we used an integrated chemical proteomics approach to develop a suite of chemical tools, including first-in-class functional inhibitors, for SLC15A4. We demonstrate that these inhibitors suppress SLC15A4-mediated endolysosomal TLR and NOD functions in a variety of human and mouse immune cells; we provide evidence of their ability to suppress inflammation in vivo and in clinical settings; and we provide insights into their mechanism of action. Our findings establish SLC15A4 as a druggable target for the treatment of autoimmune and autoinflammatory conditions.

A heterobifunctional molecule system for targeted protein acetylation in cells.

Protein acetylation is a vital biological process that regulates myriad cellular events. Despite its profound effects on protein function, there are limited research tools to dynamically and selectively regulate protein acetylation. To address this, we developed an acetylation tagging system, called AceTAG, to target proteins for chemically induced acetylation directly in live cells. AceTAG uses heterobifunctional molecules composed of a ligand for the lysine acetyltransferase p300/CBP and a FKBP12F36V ligand. Target proteins are genetically tagged with FKBP12F36V and brought in proximity with p300/CBP by AceTAG molecules to subsequently undergo protein-specific acetylation. Targeted acetylation of proteins in cells using AceTAG is selective, rapid, and can be modulated in a dose-dependent fashion, enabling controlled investigations of acetylated protein targets directly in cells. In this protocol, we focus on (1) generation of AceTAG constructs and cell lines, (2) in vitro characterization of AceTAG mediated ternary complex formation and cellular target engagement studies; and (3) in situ characterization of AceTAG induced acetylation of targeted proteins by immunoblotting and quantitative proteomics. The robust procedures described herein should enable the use of AceTAG to explore the roles of acetylation for a variety of protein targets.

Chemoproteomic mapping of human milk oligosaccharide (HMO) interactions in cells.

Human milk oligosaccharides (HMOs) are a family of unconjugated soluble glycans found in human breast milk that exhibit a myriad of biological activity. While recent studies have uncovered numerous biological functions for HMOs (antimicrobial, anti-inflammatory & probiotic properties), the receptors and protein binding partners involved in these processes are not well characterized. This can be attributed largely in part to the low affinity and transient nature of soluble glycan-protein interactions, precluding the use of traditional characterization techniques to survey binding partners in live cells. Here, we present the use of synthetic photoactivatable HMO probes to capture, enrich and identify HMO protein targets in live cells using mass spectrometry-based chemoproteomics. Following initial validation studies using purified lectins, we profiled the targets of HMO probes in live mouse macrophages. Using this strategy, we mapped hundreds of HMO binding partners across multiple cellular compartments, including many known glycan-binding proteins as well as numerous proteins previously not known to bind glycans. We expect our findings to inform future investigations of the diverse roles of how HMOs may regulate protein function.

Enhanced activity of Alzheimer disease-associated variant of protein kinase Cα drives cognitive decline in a mouse model.

Exquisitely tuned activity of protein kinase C (PKC) isozymes is essential to maintaining cellular homeostasis. Whereas loss-of-function mutations are generally associated with cancer, gain-of-function variants in one isozyme, PKCα, are associated with Alzheimer's disease (AD). Here we show that the enhanced activity of one variant, PKCα M489V, is sufficient to rewire the brain phosphoproteome, drive synaptic degeneration, and impair cognition in a mouse model. This variant causes a modest 30% increase in catalytic activity without altering on/off activation dynamics or stability, underscoring that enhanced catalytic activity is sufficient to drive the biochemical, cellular, and ultimately cognitive effects observed. Analysis of hippocampal neurons from PKCα M489V mice reveals enhanced amyloid-ß-induced synaptic depression and reduced spine density compared to wild-type mice. Behavioral studies reveal that this mutation alone is sufficient to impair cognition, and, when coupled to a mouse model of AD, further accelerates cognitive decline. The druggability of protein kinases positions PKCα as a promising therapeutic target in AD.

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells.

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein-coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase-dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal-regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal-regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression.

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.

A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor.

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

Heat shock protein 27 activity is linked to endothelial barrier recovery after proinflammatory GPCR-induced disruption.

Vascular inflammation causes endothelial barrier disruption and tissue edema. Several inflammatory mediators act through G protein­coupled receptors (GPCRs), including protease-activated receptor-1 (PAR1), to elicit inflammatory responses. The activation of PAR1 by its ligand thrombin stimulates proinflammatory, p38 mitogen-activated protein kinase (MAPK) signaling that promotes endothelial barrier disruption. Through mass spectrometry phosphoproteomics, we identified heat shock protein 27 (HSP27), which exists as a large oligomer that binds to actin, as a promising candidate for the p38-mediated regulation of barrier integrity. Depletion of HSP27 by siRNA enhanced endothelial cell barrier permeability and slowed recovery after thrombin stimulation. We further showed that two effector kinases of p38 MAPK, MAPKAPK2 (MK2) and MAPKAPK3 (MK3), differentially phosphorylated HSP27 at Ser15, Ser78, and Ser82. Whereas inhibition of thrombin-stimulated p38 activation blocked HSP27 phosphorylation at all three sites, inhibition of MK2 reduced the phosphorylation of only Ser15 and Ser78. Inhibition of both MK2 and MK3 was necessary to attenuate Ser82 phosphorylation. Thrombin-stimulated p38-MK2-MK3 signaling induced HSP27 oligomer disassembly. However, a phosphorylation-deficient mutant of HSP27 exhibited defective oligomer disassembly and altered the dynamics of barrier recovery after thrombin stimulation. Moreover, blocking HSP27 oligomer reassembly with the small-molecule inhibitor J2 enhanced endothelial barrier permeability in vitro and vascular leakage in vivo in response to PAR1 activation. These studies reveal the distinct regulation of HSP27 phosphorylation and function induced by the GPCR-stimulated p38-MK2-MK3 signaling axis that controls the dynamics of endothelial barrier recovery in vitro and vascular leakage in vivo.

Targeted Protein Acetylation in Cells Using Heterobifunctional Molecules.

Protein acetylation is a central event in orchestrating diverse cellular processes. However, current strategies to investigate protein acetylation in cells are often nonspecific or lack temporal and magnitude control. Here, we developed an acetylation tagging system, AceTAG, to induce acetylation of targeted proteins. The AceTAG system utilizes bifunctional molecules to direct the lysine acetyltransferase p300/CBP to proteins fused with the small protein tag FKBP12F36V, resulting in their induced acetylation. Using AceTAG, we induced targeted acetylation of a diverse array of proteins in cells, specifically histone H3.3, the NF-κB subunit p65/RelA, and the tumor suppressor p53. We demonstrate that targeted acetylation with the AceTAG system is rapid, selective, reversible and can be controlled in a dose-dependent fashion. AceTAG represents a useful strategy to modulate protein acetylation and should enable the exploration of targeted acetylation in basic biological and therapeutic contexts.

mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif.

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCßII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

CLK1 reorganizes the splicing factor U1-70K for early spliceosomal protein assembly.

Early spliceosome assembly requires phosphorylation of U1-70K, a constituent of the U1 small nuclear ribonucleoprotein (snRNP), but it is unclear which sites are phosphorylated, and by what enzyme, and how such modification regulates function. By profiling the proteome, we found that the Cdc2-like kinase 1 (CLK1) phosphorylates Ser-226 in the C terminus of U1-70K. This releases U1-70K from subnuclear granules facilitating interaction with U1 snRNP and the serine-arginine (SR) protein SRSF1, critical steps in establishing the 5' splice site. CLK1 breaks contacts between the C terminus and the RNA recognition motif (RRM) in U1-70K releasing the RRM to bind SRSF1. This reorganization also permits stable interactions between U1-70K and several proteins associated with U1 snRNP. Nuclear induction of the SR protein kinase 1 (SRPK1) facilitates CLK1 dissociation from U1-70K, recycling the kinase for catalysis. These studies demonstrate that CLK1 plays a vital, signal-dependent role in early spliceosomal protein assembly by contouring U1-70K for protein-protein multitasking.

Disruption of innate defense responses by endoglycosidase HPSE promotes cell survival.

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we followed heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late-stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.

Mortality Risk Profiling of Staphylococcus aureus Bacteremia by Multi-omic Serum Analysis Reveals Early Predictive and Pathogenic Signatures.

Staphylococcus aureus bacteremia (SaB) causes significant disease in humans, carrying mortality rates of ∼25%. The ability to rapidly predict SaB patient responses and guide personalized treatment regimens could reduce mortality. Here, we present a resource of SaB prognostic biomarkers. Integrating proteomic and metabolomic techniques enabled the identification of >10,000 features from >200 serum samples collected upon clinical presentation. We interrogated the complexity of serum using multiple computational strategies, which provided a comprehensive view of the early host response to infection. Our biomarkers exceed the predictive capabilities of those previously reported, particularly when used in combination. Last, we validated the biological contribution of mortality-associated pathways using a murine model of SaB. Our findings represent a starting point for the development of a prognostic test for identifying high-risk patients at a time early enough to trigger intensive monitoring and interventions.

Molecular dissection of Chagas induced cardiomyopathy reveals central disease associated and druggable signaling pathways.

Chagas disease, the clinical presentation of T. cruzi infection, is a major human health concern. While the acute phase of Chagas disease is typically asymptomatic and self-resolving, chronically infected individuals suffer numerous sequelae later in life. Cardiomyopathies in particular are the most severe consequence of chronic Chagas disease and cannot be reversed solely by parasite load reduction. To prioritize new therapeutic targets, we unbiasedly interrogated the host signaling events in heart tissues isolated from a Chagas disease mouse model using quantitative, multiplexed proteomics. We defined the host response to infection at both the proteome and phospho-proteome levels. The proteome showed an increase in the immune response and a strong repression of several mitochondrial proteins. Complementing the proteome studies, the phospho-proteomic survey found an abundance of phospho-site alterations in plasma membrane and cytoskeletal proteins. Bioinformatic analysis of kinase activity provided substantial evidence for the activation of NDRG2 and JNK/p38 kinases during Chagas disease. A significant activation of DYRK2 and AMPKA2 and the inhibition of casein family kinases were also predicted. We concluded our analyses by linking the diseased heart proteome profile to known therapeutic interventions, uncovering a potential to target mitochondrial proteins, secreted immune effectors and core kinases for the treatment of chronic Chagas disease. Together, this study provides molecular insight into host proteome and phospho-proteome responses to T. cruzi infection in the heart for the first time, highlighting pathways that can be further validated for functional contributions to disease and suitability as drug targets.

Exposure of Mycobacterium abscessus to Environmental Stress and Clinically Used Antibiotics Reveals Common Proteome Response among Pathogenic Mycobacteria.

Mycobacterium abscessus subsp. abscessus (MAB) is a clinically important nontuberculous mycobacterium (NTM) causing pulmonary infection in patients such as cystic fibrosis and bronchiectasis. MAB is naturally resistant to the majority of available antibiotics. In attempts to identify the fundamental response of MAB to aerobic, anaerobic, and biofilm conditions (as it is encountered in patients) and during exposure to antibiotics, we studied bacterial proteome using tandem mass tag mass spectrometry sequencing. Numerous de novo synthesized proteins belonging to diverse metabolic pathways were found in anaerobic and biofilm conditions, including glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, nitrogen metabolism, and glyoxylate and dicarboxylate metabolism. Upon exposure to amikacin and linezolid under stress environments, MAB displayed metabolic enrichment for glycerophospholipid metabolism and oxidative phosphorylation. By comparing proteomes of two significant NTMs, MAB and M. avium subsp. hominissuis, we found highly synthesized shared enzymes of oxidative phosphorylation, TCA cycle, glycolysis/gluconeogenesis, glyoxylate/dicarboxylate, nitrogen metabolism, peptidoglycan biosynthesis, and glycerophospholipid/glycerolipid metabolism. The activation of peptidoglycan and fatty acid biosynthesis pathways indicates the attempt of bacteria to modify the cell wall, influencing the susceptibility to antibiotics. This study establishes global changes in the synthesis of enzymes promoting the metabolic shift and enhancing the pathogen resistance to antibiotics within different environments.

Phosphoproteomic analysis of protease-activated receptor-1 biased signaling reveals unique modulators of endothelial barrier function.

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.

Organ-level protein networks as a reference for the host effects of the microbiome.

Connections between the microbiome and health are rapidly emerging in a wide range of diseases. However, a detailed mechanistic understanding of how different microbial communities are influencing their hosts is often lacking. One method researchers have used to understand these effects are germ-free (GF) mouse models. Differences found within the organ systems of these model organisms may highlight generalizable mechanisms that microbiome dysbioses have throughout the host. Here, we applied multiplexed, quantitative proteomics on the brains, spleens, hearts, small intestines, and colons of conventionally raised and GF mice, identifying associations to colonization state in over 7000 proteins. Highly ranked associations were constructed into protein-protein interaction networks and visualized onto an interactive 3D mouse model for user-guided exploration. These results act as a resource for microbiome researchers hoping to identify host effects of microbiome colonization on a given organ of interest. Our results include validation of previously reported effects in xenobiotic metabolism, the innate immune system, and glutamate-associated proteins while simultaneously providing organism-wide context. We highlight organism-wide differences in mitochondrial proteins including consistent increases in NNT, a mitochondrial protein with essential roles in influencing levels of NADH and NADPH, in all analyzed organs of conventional mice. Our networks also reveal new associations for further exploration, including protease responses in the spleen, high-density lipoproteins in the heart, and glutamatergic signaling in the brain. In total, our study provides a resource for microbiome researchers through detailed tables and visualization of the protein-level effects of microbial colonization on several organ systems.

A Hetero-Multimeric Chitinase-Containing Plasmodium falciparum and Plasmodium gallinaceum Ookinete-Secreted Protein Complex Involved in Mosquito Midgut Invasion.

Malaria parasites are transmitted by Anopheles mosquitoes. During its life cycle in the mosquito vector the Plasmodium ookinete escapes the proteolytic milieu of the post-blood meal midgut by traversing the midgut wall. This process requires penetration of the chitin-containing peritrophic matrix lining the midgut epithelium, which depends in part on ookinete-secreted chitinases. Plasmodium falciparum ookinetes have one chitinase (PfCHT1), whereas ookinetes of the avian-infecting parasite, P. gallinaceum, have two, a long and a short form, PgCHT1 and PgCHT2, respectively. Published data indicates that PgCHT2 forms a high molecular weight (HMW) reduction-sensitive complex; and one binding partner is the ookinete-produced von Willebrand A-domain-containing protein, WARP. Size exclusion chromatography data reported here show that P. gallinaceum PgCHT2 and its ortholog, P. falciparum PfCHT1 are covalently-linked components of a HMW chitinase-containing complex (> 1,300 kDa). Mass spectrometry of ookinete-secreted proteins isolated using a new chitin bead pull-down method identified chitinase-associated proteins in P. falciparum and P. gallinaceum ookinete-conditioned culture media. Mass spectrometry of this complex showed the presence of several micronemal proteins including von Willebrand factor A domain-related protein (WARP), ookinete surface enolase, and secreted ookinete adhesive protein (SOAP). To test the hypothesis that ookinete-produced PfCHT1 can form a high molecular homo-multimer or, alternatively, interacts with P. berghei ookinete-produced proteins to produce an HMW hetero-multimer, we created chimeric P. berghei parasites expressing PfCHT1 to replace PbCHT1, enabling the production of large numbers of PfCHT1-expressing ookinetes. We show that chimeric P. berghei ookinetes express monomeric PfCHT1, but a HMW complex containing PfCHT1 is not present. A better understanding of the chitinase-containing HMW complex may enhance development of next-generation vaccines or drugs that target malaria transmission stages.