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Mass spectrometry-guided molecular modeling predicts the interaction interface for a camelid single-domain antibody targeting the Plasmodium falciparum circumsporozoite protein’s C-terminal domain

ABSTRACT: To improve malaria diagnostics novel detector systems with high specificity and sensitivity are required. The malarial antigen PfCSP-Cext was selected to generate the camelid single domain antibody sdAbCSP1, which is intended to be used as capture device for detecting PfCSP-Cext in serum of acutely infected indivduals. To characterize the sdAbCSP1 functionality the 3D structure of the sdAbCSP1 – PfCSP-Cext complex was modeled in-silico. Then, the recombinantly expressed proteins sdAbCSP1, PfCSP-Cext, and the sdAbCSP1 – PfCSP-Cext complex were subjected to limited proteolysis for epitope mapping and paratope mapping. Mass spectrometry confirmed the interaction partial surfaces on sdAbCSP1 and on PfCSP-Cext. Moreover, binding strength of the sdAbCSP1 – PfCSP-Cext complex was determined by ITEM-TWO analysis and by isothermal titration calorimetry (ITC).

INSTRUMENT(S): Synapt MS

ORGANISM(S): Lama

SUBMITTER: Michael Kreutzer

LAB HEAD: Michael O. Glocker

PROVIDER: PXD051302 | Pride | 2024-08-28

REPOSITORIES: Pride

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Source:

			Action	DRS
	DB_PCR_pep_20221117.fasta	Fasta
	F417228.dat	Other
	F417228.mgf	Mgf
	NbCSP_1_PfaCSP_Cext_complex_GluC_digest_24h_pepmix_MS_01.raw.zip	Raw
	NbCSP_1_PfaCSP_Cext_complex_GluC_digest_72h_LC_MS_01.raw.zip	Raw

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Project description:The cost of Carica papaya production through seed-based propagation is increased by sex segregation, making in vitro techniques a more appealing option for clonal propagation. Inducing embryogenic callus with 2,4-dichlorophenoxyacetic acid (2,4-D) hold the potential to large-scale cloning, although the molecular mechanisms underlying this process are still not well understood. In this study, we performed a temporal analysis in the proteome of C. papaya callus to identify the key players involved in embryogenic differentiation. Mature zygotic embryos were used as explants and treated with 20 μM 2,4-D to induce embryogenic callus. Total proteins were extracted at 0, 7, 14, and 21 days (T0, T7, T14, and T21), and 1407 proteins were identified using bottom-up proteomic approach. Comparative proteomics revealed 957 differentially accumulated proteins (DAPs) (p<0.05 and log2FC >0.585 or <-0.585) in at least one comparison between the analyzed induction times points. The clustering analysis revealed four clusters with distinct patterns of protein accumulation throughout the embryogenic callus induction treatment. The cluster 1 contains 386 DAPs that accumulated at all analyzed times after treatment with 2,4-D. In contrast, cluster 2 contains 165 DAPs that decrease in abundance during the induction. The cluster 3 contains 251 proteins that are most abundant just after the start of incubation in 2,4-D (T7) and cluster 4 grouped 155 proteins that accumulate after callus formation. Functional analysis revealed that proteins involved with reserve storage and seed maturation were more abundant in the explant at T0 and decreased as callus formation progressed. Biological processes involving carbohydrate and amino acid metabolism, aerobic respiration, and protein catabolic processes were enriched after induction treatment. Regulatory proteins, including histone deacetylase (HDT3) and argonaute 1, were more abundant after the start of induction treatment with 2,4-D, suggesting their role in acquisition of embryogenic competence. Predicted protein-protein networks revealed the regulatory role of proteins 14.3.3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation, hormone response, and SAM metabolism. Our findings emphasize the modulation of the proteome at different stages during embryogenic callus initiation and identify regulatory proteins that might be involved with the activation of this process.

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Mass spectrometry-guided molecular modeling predicts the interaction interface for a camelid single-domain antibody targeting the Plasmodium falciparum circumsporozoite protein’s C-terminal domain

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