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Phosphoproteomic profiling of silk-producing glands of the spider Triconephila clavipes

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Caroline Lacerra De Souza
Total Authors: 1
Document type: Master's Dissertation
Press: Rio Claro. 2021-06-28.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Rio Claro
Defense date:
Advisor: Mario Sergio Palma; Jose Roberto Aparecido dos Santos Pinto

Despite the great interest in the mechanical-elastic properties of the silk fibers produced by spiders, aiming their use in biomedical and biotechnological applications due to their properties of resistance, elasticity and biocompatibility, little is known about the details of the silk-producing glands and the spinning process that takes place for the production of the fibers. Studies have shown that the silk producing glands, present several proteins related to the involvement in the secretion of silk proteins, transport, regulation of proteolytic activities and preservation of silk proteins, against oxidative stress and degradation, throughout the spinning process; in addition, studies have also demonstrated in the silk web fibers, of general action toxins-like and neurotoxins-like common in animal venom, suggesting that these proteins contribute to paralysis and prey capture. However, these studies have not considered the possible presence of post-translational modifications (PTMs) that can act over biological activity and function of these molecules. The determination of PTMs is fundamental for the processes elucidation that control cellular events, such as cell growth, division and differentiation, including the interaction between proteins. Therefore, in the present study we adopted the use of TiO2 affinity chromatography as a phosphorylation enrichment method, combined with the gel-free proteomic approach - shotgun, using advanced techniques of in-tandem mass spectrometry (µLC-ESI-micrOTOF-Q -III-CID) in order to identify the phosphoproteomic of glands profile that secrete the spider silk Trichonephila clavipes. The results obtained with the phosphoproteomic analysis of the set of silk producing glands of the T. clavipes spider, demonstrateded the identification of 1316 phosphorylated proteins, of which 155 were identified in the aggregated gland, 580 in the major ampulate, 143 in the flagelliform and 438 in the minor ampulate. These phosphoproteins were classified according to their general function and subdivided into seven different groups of proteins: (I) structural proteins that make up the fibers of silk - the spidroins; (II) proteins related to the transport of ions and oxygen in the glands to keep the spidroins stability; (III) proteins related to the folding/shaping and modification of spidroins; (IV) proteins related to the preservation of spidroins against oxidative stress; (V) proteins related to the preservation of the fibrillar characteristics of spidroins in the environment; (VI) housekeeping proteins and (vii) proteins related to prey capture and pre-digestion. For the last group, 284 toxin, neurotoxins, proteolytic enzymes, and defensins-like proteins, in their phosphorylated form were identified. The functional enrichment of these proteins allowed us to evaluate the contribution of their functions to the physicochemical characteristics of structural silk proteins, as well as the interaction between the functional groups of the listed proteins involved with the silk spinning process; And also the influence on the toxic activity of the toxins. The results also made it possible to identify a series of phosphorylated peptide sequences that did not present identification results with proteins deposited in the data banks and were obtained using the sequencing approach de novo and subjected to a sequence alignment using the BLASTP tool. The insect-toxicity assay carried out at the end of the study allowed us to verify the toxicity of the crude phosphorylated extract of the major ampulate and aggregated glands, which showed the lethal dose values LD50 = 28.40 ng/mg of insect (A. mellifera) for the major ampulate gland and LD50 = 23.07 ng/mg of insect (A. mellifera) for the aggregated gland. Thus, we demonstrate that the combination of using methods based on affinity chromatography, such as the strategy of enriching proteins / phosphorylated peptides, and the use of a proteomic approach with studies of PTMs proved to be an efficient strategy in the investigation of complex samples, like the silk-producing glands in the spider web. A study with post-translational modifications of proteins present in the spider producing glands of the spider T. clavipes, as demonstrated in the present work, enabled us to obtain biological and chemical information on the characteristics of these molecules, contributing to a better understanding of the phosphoproteomic profile of the glands analyzed (AU)

FAPESP's process: 19/04538-1 - Profiling the phosphoproteome from Nephila clavipes spider silk-producing glands
Grantee:Caroline Lacerra de Souza
Support type: Scholarships in Brazil - Master